DE102018001586A1 - Method for detecting the amplification of a nucleic acid with improved specificity - Google Patents

Abstract

The present invention relates to a method for amplifying a nucleic acid with improved specificity using particular primer oligonucleotides and controller nucleotides, wherein in a first amplification, the controller oligonucleotides enable a strand-opening in the amplificate in a sequence-specific manner. The invention further relates to a corresponding kit for carrying out the method according to the invention.

Description

The present invention relates to a method for the amplification of nucleic acids with improved specificity.

background

The synthesis of nucleic acid chains plays a central role in biotechnology today. Methods such as PCR have significantly advanced both the research landscape and industrial application fields, such as diagnostics in medicine and the food industry. The combination of PCR with other technologies, such as sequencing, real-time detection, microarray technology, microfluidic management, etc. has contributed to the technological evolution of the basic technology and has overcome some of the barriers to basic PCR technology. Other amplification techniques, such as isothermal amplification techniques (LAMP, HDA, RPA, TMA, SDA, etc.) have also been developed. Their use was especially intended for POCT (point-of-caretesting).

Despite tremendous advances in this field, PCR plays the central role in determining the individual technological barriers of the applications.

One of the characteristics of widespread amplification methods, such as PCR, is that during the amplification process of the nucleic acid there is no control over the amplified sequence segments between both primers. Essentially, primer binding is the focus of optimizations of PCR amplification reactions. At the beginning of the PCR amplification and in its course, a more or less specific primer binding and initiation of the synthesis of main products and by-products occurs continuously. The by-products can be generated, for example, as a result of a nonspecific primer extension event in a synthesis cycle. In case of possible re-synthesis reactions, the unspecifically extended primer is read off as a template, which generally leads to the formation of a complete primer binding site. Thus, a transmission of erroneous sequence information from one synthesis cycle to the next synthesis cycle occurs, resulting in the sum of synthesis cycles not only to initial formation, but especially to the exponential multiplication of by-products.

Such side reactions may possibly lead to initial formation and exponential multiplication of fragments which interfere with the main reaction (amplification of a target sequence) or lead to interferences in subsequent steps of the analysis. Such by-products typically include primer sequences and corresponding primer sequences so that their amplification can occur in parallel with the main reaction. However, instead of a target sequence, such by-products include a different nucleic acid sequence.

Primarily, the specificity of the PCR amplification is achieved by optimizing the primer binding to target sequences. In this case, for example, additional oligonucleotides can be used, which can partially bind to the primer and thus competitively participate in primer binding to other nucleic acid chains. Such probes typically bind to a sequence portion of the primer and release a single-stranded sequence portion on the primer so that the primer can bind to the target nucleic acid with this portion and initiate a synthesis reaction. The specificity of a primer binding is thereby to be improved, since primer-template mismatches can be competitively displaced by such oligonucleotides. As a result, the specificity of the initiation of PCR reactions can usually be improved. The effect of such oligonucleotides is limited to interaction with primer sequences. Such additional oligonucleotides do not interact with the nucleic acid chain to be amplified in portions between the two primers. However, due to a molar excess of the primers, non-specific interactions of primers with templates may occur during amplification. If such an unspecific event of primer extension occurs (initiation of an exponential side reaction), then a fully functional primer binding site will result, or in the context of reverse synthesis of a complementary strand of the by-product. The presence of such a complete primer binding site in the by-product results in a loss of the competitive effect of such additional oligonucleotides on primer binding. The control of the specificity of the binding of a primer to the template by such oligonucleotides thus makes only the initiation of a side reaction less likely, but can hardly influence its exponential amplification after formation of a byproduct.

Side reactions in a PCR increase with the number of synthetic cycles performed. In some applications, up to 30 - 40 PCR cycles have to be performed, so that a sufficient amount of PCR products is accumulated. After the PCR amplification, other methods can be connected, eg detection, library production, cloning, sequencing, etc. These methods can tolerate only to a certain extent the by-products or sequence deviations produced during a PCR amplification. In general, the specificity of such methods is also influenced by the problem of side reactions during a PCR.

In general, improving the specificity of the synthesis of a amplification process with reduced generation and co-amplification of by-products other than target sequence may contribute, for example, to an improvement in diagnostic procedures.

It is therefore an object of the present invention to provide methods and components which enable the enzymatic synthesis and amplification of nucleic acid chains. The aim is to provide a novel enzymatic process and components for the synthesis of nucleic acid chains, as well as the amplification of nucleic acid chains.

A further object of the invention is to provide a combination of an amplification method with a PCR amplification, wherein the signal detection (monitoring or detection), or the amplification of target sequences, first by means of controlled amplification using controller oligonucleotide and corresponding specific primer Sets is carried out and then continued by means of PCR, in particular using PCR-specific primer.

Another object of the invention is to reduce the number of PCR synthesis cycles necessary to obtain a sufficient amount of PCR products.

Another object of the invention is to provide a method with improved specificity of the synthesis of target nucleic acid chains in an exponential amplification.

Another object of the invention is to provide means for implementing an exponential amplification process with improved specificity of the synthesis.

A further object of the invention is to provide a novel enzymatic method and components for the synthesis of nucleic acid chains, as well as the amplification of nucleic acid chains, in which a continuous (on-line) signal detection is provided, wherein the signal acquisition (Monitoring / Detection) by sequence-specific oligonucleotide probes.

These objects are achieved by a method having the features of claim 1 and a kit having the features of claim XY. Further advantageous embodiments of the invention are set forth in the dependent claims and the following description.

Description of the invention

1. A) Bereitstellung eines Nukleinsäurefragmentes umfassend eine erste Zielsequenz (eine Start-Nukleinsäurekette)
2. B) Bereitstellung eines ersten Amplifikations-Systems, umfassend:
   • • Ein erstes Primer-Oligonukleotid
   • • Ein zweites Primer-Oligonukleotid
   • • Ein Controller-Oligonukleotid
   • • Eine erste Polymerase
   • • Substrate für erste Polymerase (dNTPs) und geeigneten Puffer-Lösung
3. C) Bereitstellung eines zweiten Amplifikations-Systems, umfassend:
   • • Ein drittes Primer-Oligonukleotid
   • • Ein viertes Primer-Oligonukleotid
   • • Eine zweite Polymerase, welche eine thermostabile Polymerase umfasst
   • • Substrate für zweite Polymerase (dNTPs) und geeigneten Puffer-Lösung
   • • Ein Detektions-System
4. D) Durchführung einer ersten Amplifikation unter Verwendung der Start-Nukleinsäurekette als initialen Matrizenstrang und des ersten Amplifikationssystems, unter Erhalt eines ersten Amplifikations-Fragmetnes 1.1, umfassend die erste Zielsequenz, und umfassend ein erstes Primer-Verlängerungsprodukt und ein zweites Primer-Verlängerungsprodukt, welche einen komplementären Doppelstrang bilden können, wobei das erste Primer-Verlängerungsprodukt resultiert aus matrizenabhängigen Verlängerung eines ersten Primers mittels Polymerase unter Verwendung der Start-Nukleinsäurekette und / oder des zweiten Primer-Verlängerungsproduktes als Matrize und das das zweite Primer-Verlängerungsprodukt resultiert aus matrizenabhängigen Verlängerung eines zweiten Primers mittels Polymerase unter Verwendung des ersten Primer-Verlängeungsproduktes als Matrize, wobei das erste und das zweite Primer-Verlängerungsprodukte gegenseitig als Matrizen für die jeweilige Primer-Verlängerung dienen können und wobei beide komplementären Stränge des ersten Amplifikations-Produktes 1.1 unter Mitwirkung des Controller-Oligonukleotides zumindest teilweise in einzelsträngige Form überführt werden können, so dass erneute Primer-Bindung an jeweils komplementäre Seqmente der synthetisierten Primer-Verlängerungsprodutke möglich ist und die verwendeten Reaktionsbedingen der ersten Amplifikation mindestens einen Temperatur-Schritt umfassen, welcher Hybridisierung und matrizenabhängige Primer-Verlängerung von beiden Primern des ersten Amplifikations-Systems zuläßt und mindestens einen Temperatur-Schritt umfassen, bei welchen das erste Primer-Verlängerungsprodukt vom zweiten Primer-Verlängerungsprodukt unter Mitwirkung des Controller-Oligonukleotides getrennt wird, wobei die verwendeten Reaktionsbedingungen keine spontante Trennung des ersten Primer-Verlängerungsproduktes vom zweiten Primer-Verlängerungsprodukt in Abwesenheit des Controller-Oligonukleotides zulassen. Wobei die erste Amplifikation so lange durchgeführt wird bis die gewünschte Menge des ersten Amplifikationsproduktes 1.1 synthetisiert wird.
5. E) Durchführung einer zweiten Amplifikation unter Verwendung von mindestens einem der beiden Stränge des ersten Amplifikations-Fragmentes 1.1 als initialer Matrizenstrang und unter Verwendung des zweiten Amplifikations-Systems unter Erhalt eines zweiten Amplifikations-Fragmenten 2.1, umfassend ein drittes Primer-Verlängerungsprodukt und ein viertes Primer-Verlängerungsprodukt, welche einen komplementären Doppelstrang bilden können, wobei beide Stränge des Amplifikations-Fragmenten 2.1 gegenseitig als Matrizen bei Primer-Verlängerung dienen können, wobei die verwendeten Reaktionsbedingungen der zweiten Amplifikation mindestens in einem Temperatur-Schritt eine Hybridisierung der beiden Primer des zweiten Amplifikations-Systems und eine matrizenabhängige Primer-Verlängerung des jeweils an komplementäre Bereiche gebundenen Primers des zweiten Amplifikations-Systems zulassen und mindestens in einem weiteren Temperatur-Schritt eine Trennung eines Doppelstranges zulassen, umfassend ein drittes Primer-Verlängerungsprodukt und ein viertes Primer-Verlängerungsprodukt, wobei das dritte und das vierte Primer-Verlängerungsprodukte in einzelstränige Form überführt werden, so dass eine erneute Primer-Bindung und Verlängerungs stattfinden kann. Wobei die zweite Amplifikation so lange durchgeführt wird bis die gewünschte Menge des zweiten Amplifikationsproduktes 2.1 synthetisiert wird.

According to the present invention, a method for amplification is provided. The method comprises the steps:

1. A) Provision of a nucleic acid fragment comprising a first target sequence (a start nucleic acid chain)
2. B) providing a first amplification system comprising:
   • A first primer oligonucleotide
   • A second primer oligonucleotide
   • • A controller oligonucleotide
   • • A first polymerase
   • • Substrates for first polymerase (dNTPs) and suitable buffer solution
3. C) providing a second amplification system comprising:
   • A third primer oligonucleotide
   • A fourth primer oligonucleotide
   • A second polymerase comprising a thermostable polymerase
   • Substrates for second polymerase (dNTPs) and appropriate buffer solution
   • • A detection system
4. D) Performing a first amplification using the starting nucleic acid chain as the initial template strand and the first amplification system to obtain a first amplification fragment 1.1 comprising the first target sequence, and comprising a first primer extension product and a second primer extension product which can form a complementary duplex, wherein the first primer extension product results from template-dependent extension of a first primer by means of polymerase using the start nucleic acid chain and / or the second primer extension product as a template, and the second primer extension product results from template-dependent extension of a second primer using polymerase using the first primer extension product as template, wherein the first and second primer extension products mutually serve as templates for the respective primers Extension and can serve both complementary strands of the first amplification product 1.1 can be converted at least partially into single-stranded form with the assistance of the controller oligonucleotide, so that renewed primer binding to respectively complementary segments of the synthesized primer extension product is possible and the reaction conditions of the first amplification used comprise at least one temperature step which hybridization and template-dependent Primer extension of both primers of the first amplification system and comprising at least one temperature step in which the first primer extension product is separated from the second primer extension product with the assistance of the controller oligonucleotide, the reaction conditions used no spontaneous separation of the first Allow primer extension product from the second primer extension product in the absence of the controller oligonucleotide. Wherein the first amplification is carried out until the desired amount of the first amplification product 1.1 is synthesized.
5. E) carrying out a second amplification using at least one of the two strands of the first amplification fragment 1.1 as an initial template strand and using the second amplification system to give a second amplification fragment 2.1 comprising a third primer extension product and a fourth primer extension product which can form a complementary duplex, both strands of the amplification fragments 2.1 can serve as matrices for primer extension, wherein the reaction conditions of the second amplification used hybridization of the two primers of the second amplification system at least in one temperature step and a template-dependent primer extension of each bound to complementary regions primer of the second amplification System and allow separation of a duplex at least in a further temperature step, comprising a third primer extension product and a fourth primer extension product, wherein the third and fourth primer extension products are converted into single stranded form such that a re-primer Binding and extension can take place. Wherein the second amplification is carried out until the desired amount of the second amplification product 2.1 is synthesized.

In detail, the properties of components of the respective amplification system, the nucleic acid fragment comprising a first target sequence, and the reaction conditions used determine the course of the two amplification reactions.

In general, the first amplification takes place with the assistance of the controller oligonucleotide. In this case, the separation of the two synthesized primer extension products takes place at least in part depending on the sequence composition of the first primer extension product and its complementarity with respect to the composition of the controller oligonucleotide.

During the second amplification becomes a second amplification fragment 2.1 amplified essentially without the involvement of the controller oligonucleotides.

In the course of both amplification reactions, both desired amplification products (amplification fragment 1.1 and amplification fragment 2.1 ) as well as their intermediate stages (Intermediate products). The composition of the intermediates depends essentially on the provided components of the first and second amplification systems.

In the following, some molecular processes and the resulting products and the potential intermediates will be exemplarily and schematically explained.

• 1) eine erste Amplifikation mit den Schritten
  • 1.A) Hybridisieren eines ersten Primer-Oligonukleotids an das 3'-Segment eines als Matrize dienenden Nukleinsäurefragments (Matrizenstrang, Start-Nukleinsäurekette) einer ersten zu amplifizierenden Nukleinsäure, welche eine erste Zielsequenz umfasst, wobei das erste Primer-Oligonukleotid folgende Bereiche umfasst:
    • • einen ersten Bereich, der sequenzspezifisch an das 3' -Segment eines Matrizenstranges der ersten zu amplifizierenden Nukleinsäure binden kann und komplementär zumindest zu einem Teil der Zielsequenz ist;
    • • einen zweiten Bereich, der sich an das 5'-Ende des ersten Bereichs anschließt oder über einen Linker verbunden ist, wobei der zweite Bereich von einem Controller-Oligonukleotids gebunden werden kann und von einer für die ersten Amplifikation verwendete Polymerase unter den gewählten Reaktionsbedingungen im Wesentlich unkopiert bleibt;
  • 1.B) Verlängerung des ersten Primer-Oligonukleotids wenn hybridisiert an komplementäre Segmente einer ersten zu amplifizierenden Nukleionsäure durch die erste matrizenabhängige Polymerase unter Erhalt eines ersten Primer-Verlängerungsprodukt, welches neben dem ersten Primer-Oligonukleotid einen von Polymerase synthetisierten Bereich umfasst, der im Wesentlichen komplementär zum Matrizenstrang der ersten zu amplifizierenden Nukleinsäure ist, welcher eine erste Zielsequenz umfasst, wobei das erste Primer-Verlängerungsprodukt und der Matrizenstrang der ersten zu amplifizierenden Nukleinsäure im Wesentlichen als Doppelstrang unter verwendeten Reaktionsbedingungen der ersten Amplifikation vorliegen;
  • 1.C) Bindung eines Controller-Oligonukleotids an das erste Primer-Verlängerungsprodukt, wobei das Controller-Oligonukleotid folgende Bereiche umfasst:
    • • einen ersten Bereich, der an den zweiten Bereich des ersten Primers und / oder des ersten Primer-Verlängerungsprodukts binden kann;
    • • einen zweiten Bereich, der zum ersten Bereich des ersten Primer-Oligonukleotid und / oder des ersten Primer-Verlängerungsproduktes im Wesentlichen komplementär oder vollständig komplementär ist, und
    • • einen dritten Bereich, der zumindest zu einen Teil des von Polymerase synthetisierten Bereiches des ersten Primer-Verlängerungsprodukts im Wesentlichen komplementär oder vollständig komplementär ist;
    wobei das Controller-Oligonukleotide nicht als Matrize für eine Primerverlängerung des ersten Primer-Oligonukleotids dient, und das Controller-Oligonukleotid ein Sequenzsegment umfasst, welches zu einem Strang der ersten zu amplifizierenden Nukleinsäure komplementär oder im Wesentlichen komplementär ist, und das Controller-Oligonukleotid an den ersten Bereich des ersten Primer-Verlängerungsprodukts bindet und und Controller-Oligonukleotid an den zweiten Bereich des ersten Primer-Verlängerungsprodutkes, sowie zumindest teilweise an den synthetisierten Bereich des ersten Primer-Verlängerungsproduktes unter Verdrängung von komplementären Anteilen des Matrizenstrangs der ersten zu amplifizierenden Nukleinsäure bindet; wobei der von Polymerase synthetisierten Bereich des ersten Primer-Verlängerungsproduktes umfassend das zum zweiten Primer-Oligonukleotid komplementäres Segment des ersten Primer-Verlängerungsproduktes unter Reaktionsbedingungen einzelsträngig wird
  • 1.D) Hybridisieren eines zweiten Primer-Oligonukleotids an das einzelsträngige komplementäre Segment des ersten Primer-Verlängerungsprodukts, wobei das zweite Primer-Oligonukleotid einen Bereich umfasst, der sequenzspezifisch an den synthetisierten Bereich des ersten Primer-Verlängerungsprodukts hybridisieren kann und mindestens einen Anteil einer Zielsequenz umfasst
  • 1.E) Verlängerung des zweiten Primer-Oligonukleotids durch die erste Polymerase unter Verwendung des ersten Primer-Verlängerungsproduktes als Matrize unter Erhalt eines zweiten Primer-Verlängerungsproduktes , welches neben dem zweiten Primer-Oligonukleotid einen von Polymerase synthetisierten Bereich umfasst, der zumindest einen Anteil der ersten Zielsequenz umfasst, wobei das erste Primer-Verlängerungsprodukt und das zweite Primerverlängerungsprodukt unter Reaktionsbedingungen der ersten Amplifikation ein erstes doppelsträngiges Amplifikationsprodukt bilden; und;
  • 1.F) Ggf. Wiederholung der Schritte der ersten Amplifikation
• 2) eine zweite Amplifikation mit den Schritten (21):
  • 2.A) Hybridisieren eines dritten Primer-Oligonukleotids an das zweite Primer-Verlängerungsprodukt des ersten Amplifikationsprodukts, wobei das dritte Primer-Oligonukleotid einen ersten Bereichs umfasst, der im Wesentlichen sequenzspezifisch an ein Segment des zweiten Primer-Verlängerungsprodukt binden kann;
  • 2.B) Verlängerung des dritten Primer-Oligonukleotids durch eine zweite Polymerase unter Verwendung des zweiten Primer-Verlängerungsprodutkes als Matrize unter Erhalt eines dritten Primer-Verlängerungsprodukt (P3.1-Ext Teil 1), welches neben dem dritten Primer-Oligonukleotid einen von Polymerase synthetisierten Bereich umfasst, der ein Sequenzsegment umfasst, welches im Wesentlichen komplementär zum zweiten Primer-Verlängerungsprodukt bzw. zur ersten zu amplifizierenden Nukleinsäure oder zu einem Anteil der ersten Zielsequenz ist, wobei das zweite Primer-Verlängerungsprodukt und das dritte Primer-Verlängerungsprodukt (P3.1-Ext Teil 1) als Doppelstrang vorliegen;
  • 2C) Hybridisieren eines vierten Primer-Oligonukleotids an das erste Primer-Verlängerungsprodukts des ersten Amplifikationsprodukts, wobei das vierte Primer-Oligonukleotid einen ersten Bereichs umfasst, der im Wesentlichen sequenzspezifisch an den von Polymerase synthetisierten Bereich des ersten Primer-Verlängerungsprodukts binden kann;
  • 2D) Verlängerung des vierten Primer-Oligonukleotids durch die zweite Polymerase unter Verwendung des ersten Primer-Verlängerungsproduktes als Matrize unter Erhalt eines vierten Primer-Verlängerungsproduktes (P4.1-Ext Teil 1), welches neben dem vierten Primer-Oligonukleotid einen von Polymerase synthetisierten Bereich umfasst, der im Wesentlichen komplementär zum ersten Primer-Verlängerungsprodukt und Anteile der Zielsequenz umfasst, wobei das erste Primer-Verlängerungsprodukt und das vierte Primer-Verlängerungsprodukt (P4.1-Ext Teil 1) als Doppelstrang vorliegen;
  • 2E) Trennung des Doppelstrangs aus dem ersten Primer-Verlängerungsprodukt und dem vierten Primer-Verlängerungsprodukt (P4.1-Ext Teil 1) und des Doppelstrangs aus dem zweiten Primer-Verlängerungsprodukt und dem dritten Primer-Verlängerungsprodukt (P3.1-Ext Teil 1);
  • 2.F) Hybridisieren eines dritten Primer-Oligonukleotids an das vierte Primer-Verlängerungsprodukt (erhalten im Schritt 2D, P4.1-Ext Teil 1), wobei das dritte Primer-Oligonukleotid einen ersten Bereichs umfasst, der im Wesentlichen sequenzspezifisch an ein Segment des vierten Primer-Verlängerungsprodukt (P4.1-Ext Teil 1) binden kann;
  • 2.G) Verlängerung des dritten Primer-Oligonukleotids durch eine zweite Polymerase unter Verwendung des vierten Primer-Verlängerungsprodutkes (P4.1-Ext Teil 1) als Matrize unter Erhalt eines vollständigen dritten Primer-Verlängerungsprodukt (P3.1-Ext), wobei das dritte Primer-Verlängerungsprodukt (P3.1-Ext) und das vierte Primer-Verlängerungsprodukt (P4.1-Ext Teil 1) als Doppelstrang vorliegen;
  • 2H) Hybridisieren eines vierten Primer-Oligonukleotids an das dritte Primer-Verlängerungsprodukts (erhalten im Schritt 2B, P3.1-Ext Teil 1), wobei das vierte Primer-Oligonukleotid einen ersten Bereichs umfasst, der im Wesentlichen sequenzspezifisch an den von Polymerase synthetisierten Bereich des dritten Primer-Verlängerungsprodukts binden kann;
  • 2I) Verlängerung des vierten Primer-Oligonukleotids durch die zweite Polymerase unter Verwendung des dritten Primer-Verlängerungsproduktes (P3.1-Ext Teil 1) als Matrize unter Erhalt eines vollständigen vierten Primer-Verlängerungsproduktes (P4.1-Ext), wobei das dritte Primer-Verlängerungsprodukt (P3.1-Ext Teil 1) und das vierte Primer-Verlängerungsprodukt (P4.1-Ext) als Doppelstrang vorliegen;
  • 2J) Trennung des Doppelstrangs aus dem dritten Primer-Verlängerungsprodukt (P3.1-Ext Teil 1) und dem vollständigen vierten Primer-Verlängerungsprodukt (P4.1-Ext) und des Doppelstrangs aus dem vierten Primer-Verlängerungsprodukt (P4.1-Ext Teil 1) und dem vollständigen dritten Primer-Verlängerungsprodukt (P3.1-Ext);
  • 2.K) Hybridisieren eines dritten Primer-Oligonukleotids an das vollständige vierte Primer-Verlängerungsprodukt (erhalten im Schritt 2I, P4.1-Ext), wobei das dritte Primer-Oligonukleotid einen ersten Bereichs umfasst, der im Wesentlichen sequenzspezifisch an ein Segment des vierten Primer-Verlängerungsprodukt (P4.1-Ext) binden kann;
  • 2.L) Verlängerung des dritten Primer-Oligonukleotids durch eine zweite Polymerase unter Verwendung des vollständigen vierten Primer-Verlängerungsprodutkes (P4.1-Ext) als Matrize unter Erhalt eines vollständigen dritten Primer-Verlängerungsprodukt (P3.1-Ext), wobei das vollständige dritte Primer-Verlängerungsprodukt (P3.1-Ext) und das vollständige vierte Primer-Verlängerungsprodukt (P4.1-Ext) als Doppelstrang vorliegen;
  • 2M) Hybridisieren eines vierten Primer-Oligonukleotids an das vollständige dritte Primer-Verlängerungsprodukts (erhalten im Schritt 2G, P3.1-Ext), wobei das vierte Primer-Oligonukleotid einen ersten Bereichs umfasst, der im Wesentlichen sequenzspezifisch an den von Polymerase synthetisierten Bereich des vollständigen dritten Primer-Verlängerungsprodukts binden kann;
  • 2N) Verlängerung des vierten Primer-Oligonukleotids durch die zweite Polymerase unter Verwendung des vollständigen dritten Primer-Verlängerungsproduktes (P3.1-Ext) als Matrize unter Erhalt eines vollständigen vierten Primer-Verlängerungsproduktes (P4.1-Ext), wobei das vollständige dritte Primer-Verlängerungsprodukt (P3.1-Ext) und das vollständige vierte Primer-Verlängerungsprodukt (P4.1-Ext) als Doppelstrang vorliegen;
  • 2O) Trennung der in Schritt 2L und 2N gebildeten Doppelstränge aus dem vollständigen dritten Primer-Verlängerungsprodukt (P3.1-Ext) und dem vollständigen vierten Primer-Verlängerungsprodukt (P4.1-Ext)
  • 2.P) Ggf. Wiederholung der Schritte der zweiten Amplifikation unter Vermehrung des vollständigen dritten Primer-Verlängerungsproduktes (P3.1-Ext) und des vollständigen vierten Primer-Verlängerungsproduktes (P4.1-Ext).

wobei der Reaktionsmischung ein Detektionssystem zugegeben wird.According to a first aspect of the invention, a method for amplification is provided. The method comprises the steps:

• 1) a first amplification with the steps
  • 1.A) hybridizing a first primer oligonucleotide to the 3 'segment of a template nucleic acid fragment (template strand, starting nucleic acid chain) of a first nucleic acid to be amplified which comprises a first target sequence, wherein the first primer oligonucleotide comprises the following regions:
    • A first region which can sequence-specifically bind to the 3 'segment of a template strand of the first nucleic acid to be amplified and is complementary to at least a portion of the target sequence;
    • A second region connected to the 5 'end of the first region or connected via a linker, which second region can be bound by a controller oligonucleotide and by a polymerase used for the first amplification under the selected reaction conditions in the Essentially remains uncopied;
  • 1.B) extension of the first primer oligonucleotide when hybridized to complementary segments of a first nucleic acid to be amplified by the first template-dependent polymerase to yield a first primer extension product comprising, in addition to the first primer oligonucleotide, a polymerase-synthesized region complementary to the template strand of the first nucleic acid to be amplified which comprises a first target sequence, wherein the first primer extension product and the template strand of the first nucleic acid to be amplified are present substantially as a double strand under used reaction conditions of the first amplification;
  • 1.C) binding a controller oligonucleotide to the first primer extension product, wherein the controller oligonucleotide comprises the following ranges:
    • A first region capable of binding to the second region of the first primer and / or the first primer extension product;
    • A second region substantially complementary or fully complementary to the first region of the first primer oligonucleotide and / or the first primer extension product, and
    • A third region that is substantially complementary or fully complementary to at least a portion of the polymerase-synthesized portion of the first primer extension product;
    wherein the controller oligonucleotide does not serve as a template for primer extension of the first primer oligonucleotide, and the controller oligonucleotide comprises a sequence segment that is complementary or substantially complementary to a strand of the first nucleic acid to be amplified, and the controller oligonucleotide to the binding the first region of the first primer extension product and binding the controller oligonucleotide to the second region of the first primer extension product as well as at least partially to the synthesized region of the first primer extension product displacing complementary portions of the template strand of the first nucleic acid to be amplified; wherein the polymerase-synthesized portion of the first primer extension product comprising the second primer-oligonucleotide-complementary segment of the first primer extension product becomes single-stranded under reaction conditions
  • 1.D) hybridizing a second primer oligonucleotide to the single-stranded complementary segment of the first primer extension product, wherein the second primer oligonucleotide comprises a region capable of hybridizing sequence-specifically to the synthesized region of the first primer extension product and at least a portion of a target sequence includes
  • 1.E) extension of the second primer oligonucleotide by the first polymerase using the first primer extension product as a template to yield a second primer extension product which comprises, in addition to the second primer oligonucleotide, a polymerase-synthesized region comprising at least a portion of the first target sequence, wherein the first primer extension product and the second primer extension product form a first double-stranded amplification product under reaction conditions of the first amplification; and;
  • 1.F) If necessary Repetition of the steps of the first amplification
• 2) a second amplification with the steps ( 21 ):
  • 2.A) hybridizing a third primer oligonucleotide to the second primer extension product of the first amplification product, wherein the third primer oligonucleotide comprises a first region capable of substantially sequence-specific binding to a segment of the second primer extension product;
  • 2.B) extension of the third primer oligonucleotide by a second polymerase using the second primer extension product as a template to give a third primer extension product ( P3.1 -Ext part 1 ) comprising, in addition to the third primer oligonucleotide, a polymerase synthesized region comprising a sequence segment substantially complementary to the second primer extension product or to the first nucleic acid to be amplified or to a portion of the first target sequence, the second primer Extension product and the third primer extension product ( P3.1 -Ext part 1 ) are present as a double strand;
  • 2C) hybridizing a fourth primer oligonucleotide to the first primer extension product of the first amplification product, the fourth primer oligonucleotide comprising a first region capable of substantially sequence-specific binding to the polymerase-synthesized region of the first primer extension product;
  • 2D) extension of the fourth primer oligonucleotide by the second polymerase using the first primer extension product as template to give a fourth primer extension product ( P4.1 -Ext part 1 ) comprising, in addition to the fourth primer oligonucleotide, a polymerase-synthesized region substantially complementary to the first primer extension product and portions of the target sequence, wherein the first primer extension product and the fourth primer extension product ( P4.1 -Ext part 1 ) are present as a double strand;
  • 2E) separation of the duplex from the first primer extension product and the fourth primer extension product ( P4.1 -Ext part 1 ) and the double strand from the second primer extension product and the third primer extension product ( P3.1 -Ext part 1 );
  • 2.F) hybridizing a third primer oligonucleotide to the fourth primer extension product (obtained in step 2D . P4.1 -Ext part 1 ), wherein the third primer oligonucleotide comprises a first region which is substantially sequence-specific to a segment of the fourth primer extension product ( P4.1 -Ext part 1 ) can bind;
  • 2.G) extension of the third primer oligonucleotide by a second polymerase using the fourth primer extension product ( P4.1 -Ext part 1 ) as a template to give a complete third primer extension product ( P3.1 Text), the third primer extension product ( P3.1 -Ext) and the fourth primer extension product ( P4.1 -Ext part 1 ) are present as a double strand;
  • 2H) hybridizing a fourth primer oligonucleotide to the third primer extension product (obtained in step 2 B . P3.1 -Ext part 1 ), wherein the fourth primer oligonucleotide comprises a first region capable of substantially sequence-specific binding to the polymerase-synthesized region of the third primer extension product;
  • 2I) extension of the fourth primer oligonucleotide by the second polymerase using the third primer extension product ( P3.1 -Ext part 1 ) as a template to give a complete fourth primer extension product ( P4 .1-Ext), wherein the third primer extension product ( P3.1 -Ext part 1 ) and the fourth primer extension product ( P4.1 -Ext) as a double strand;
  • 2J) separation of the duplex from the third primer extension product ( P3.1 -Ext part 1 ) and the complete fourth primer extension product ( P4.1 -Ext) and the double strand from the fourth primer extension product ( P4.1 -Ext part 1 ) and the complete third primer extension product ( P3.1 -Ext);
  • 2.K) hybridizing a third primer oligonucleotide to the complete fourth primer extension product (obtained in step 2I . P4 .1-Ext), wherein the third primer oligonucleotide comprises a first region substantially sequence-specific to a segment of the fourth primer extension product (FIG. P4 1-Ext);
  • 2.L) extension of the third primer oligonucleotide by a second polymerase using the complete fourth primer extension product ( P4.1 -Ext) as template to give a complete third primer extension product ( P3.1 Text), with the complete third primer extension product ( P3.1 Text) and the complete fourth primer extension product ( P4.1 -Ext) as a double strand;
  • 2M) hybridizing a fourth primer oligonucleotide to the complete third primer extension product (obtained in step 2G . P3.1 ), Wherein the fourth primer oligonucleotide comprises a first region capable of substantially sequence-specific binding to the polymerase-synthesized region of the complete third primer extension product;
  • 2N) extension of the fourth primer oligonucleotide by the second polymerase using the complete third primer extension product ( P3.1 -Ext) as template to give a complete fourth primer extension product ( P4.1 Text), with the complete third primer extension product ( P3.1 Text) and the complete fourth primer extension product ( P4.1 -Ext) as a double strand;
  • 2O) Separation of in step 2L and 2N formed double strands from the complete third primer extension product ( P3.1 Text) and the complete fourth primer extension product ( P4.1 -ext)
  • 2.P) If necessary Repetition of the steps of the second amplification with multiplication of the complete third primer extension product ( P3.1 -Ext) and the complete fourth primer extension product ( P4.1 -Ext).

wherein a detection system is added to the reaction mixture.

The amount of yields of individual products and intermediates depends on several factors. For example, higher concentrations of individual primers generally favor the yields of products / intermediates. Furthermore, the formation of products / intermediates can be influenced by reaction temperature and binding affinity of individual reactants to each other (affinity of binding of individual primers to their complementary primer binding sites within template strands): in general, for example, longer oligonucleotides bind better at higher temperatures than shorter oligonucleotides Furthermore, CG content may play a role in complementary sequence segments: CG-richer sequences also bind more strongly at higher temperatures than AT-rich sequences. Furthermore, modifications such as MGB or 2-amino-dA or LNA can increase the binding strength of primers to their respective complementary segments, which also results in preferential binding of primers at higher temperatures.

It can be deduced from this that the design of sequence segments of individual primer sequences can influence the yields of certain products / intermediates and thus influence their accumulation during amplification.

1. a) eine erste Amplifikation mit den Schritten:
   • - Bereitstellung einer Start-Nukleinsäure 1.1, welche eine Zielsequenz umfasst
   • - Hybridisieren eines ersten Primer-Oligonukleotids (P1.1) an eine zu amplifizierenden Nukleinsäure mit einer Zielsequenz (entweder Start-Nukleinsäure 1.1 oder P2.1-Ext), wobei das erste Primer-Oligonukleotid (P1.1) folgende Bereiche umfasst:
     • • einen ersten Bereich (P1.1.1), der sequenzspezifischen an einen Bereich der zu amplifizierenden Nukleinsäure (P2.1 E1) binden kann,
     • • einen zweiten Bereich (P1.1.2), der sich an das 5'-Ende des ersten Bereichs anschließt oder über einen Linker verbunden ist, wobei der zweite Bereich von einem Controller-Oligonukleotid gebunden werden kann und für die verwendete Polymerase unter den gewählten Reaktionsbedingungen im Wesentlich unkopiert bleibt;
   • - Verlängerung des ersten Primer-Oligonukleotids (P1.1) durch eine erste Polymerase unter Erhalt eines ersten Primer-Verlängerungsprodukt (P1.1-Ext), welches neben dem ersten Primer-Oligonukleotid (P1.1) einen synthetisierten Bereich umfasst (P1.1E1 bis P1.1E4), der im Wesentlichen komplementär zu der zu amplifizierenden Nukleinsäure oder zur Zielsequenz ist, wobei das erste Primer-Verlängerungsprodukt und die zu amplifizierenden Nukleinsäure als Doppelstrang vorliegen;
   • - Bindung eines Controller-Oligonukleotids (C1.2) an das erste Primer-Verlängerungsprodukt (P1.1-Ext), wobei das Controller-Oligonukleotid (C1.2) folgende Bereiche umfasst:
     • • einen ersten Bereich (C1.2.1), der an den zweiten Bereich (Überhang) des ersten Primer-Verlängerungsprodukts (P1.1E6) binden kann,
     • • einen zweiten Bereich (C1.2.2), der zum ersten Bereich des ersten Primer-Oligonukleotid (P1.1.1) komplementär ist, und
     • • einen dritten Bereich (C1.2.3), der zumindest zu einen Teil des synthetisierten Bereiches des ersten Primer-Verlängerungsprodukts (P1.1E4) im Wesentlichen komplementär ist; und
     wobei das Controller-Oligonukleotide (C1.1) nicht als Matrize für eine Primerverlängerung des ersten Primer-Oligonukleotids (P1.1) dient, und das erste Controller-Oligonukleotid (C1.1) an den ersten Primer-Verlängerungsprodukt (P1.1E6, P1.1E5, P1.1E4) unter Verdrängung des zu diesem Bereich komplementären Bereich (P2.1 E1, P2.1E2) der zu amplifizierenden Nukleinsäure bindet;
   • - Hybridisieren eines zweiten Primer-Oligonukleotids (P1.1) an das erste Primer-Verlängerungsprodukt, wobei das zweite Primer-Oligonukleotid (P2.1) einen Bereich umfasst (P2.1.1), der sequenzspezifisch an den synthetisierten Bereich des ersten Primer-Verlängerungsprodukts (P1.1E1) binden kann,
   • - Verlängerung des zweiten Primer-Oligonukleotids (P.2.1) durch die erste Polymerase unter Erhalt eines zweiten Primer-Verlängerungsproduktes (P2.1-Ext), welches neben dem zweiten Primer-Oligonukleotid (P2.1) einen synthetisierten Bereich (P2.1E4 bis P2.1E1) umfasst, der im Wesentlichen identisch zu der zu amplifizierenden Nukleinsäure oder zur Zielsequenz ist, wobei das erste Primer-Verlängerungsprodukt (P1.1-Ext) und das zweite Primerverlängerungsprodukt (P2.1) ein erstes doppelsträngiges Amplifikationsprodukt bilden; und
   • - Schritte der ersten Amplifikation wiederholt werden, bis die gewünschte Menge an ersten Amplifikationsprodukten synthetisiert wurde.
2. b) eine zweite Amplifikation mit den Schritten:
   • - Hybridisieren eines dritten Primer-Oligonukleotids (P3.2) an das zweite und / oder das vierte Primer-Verlängerungsprodukt (P1.1-Ext. oder P4.1-Ext), wobei der dritte Primer-Oligonukleotid (P3.2) einen ersten Bereichs (P3.1.1) umfasst, der sequenzspezifisch an einen Bereich des zweiten Primer-Verlängerungsprodukt (P2.1E1) und des vierten Primer-Verlängerungsprodukts (P4.1 E2) binden kann,
   • - Verlängerung des dritten Primer-Oligonukleotids (P3.2) durch eine zweite Polymerase unter Erhalt eines dritten Primer-Verlängerungsprodukt (P3.2-Ext), welches neben dem dritten Primer-Oligonukleotid (P3.2) einen synthetisierten Bereich (P3.1 E5 bis P3.1E2 bzw. P3.1E5 bis P3.1E1) umfasst, der im Wesentlichen komplementär zum zweiten Primer-Verlängerungsprodukt (P2.1-Ext) oder zur Zielsequenz ist, wobei das zweite Primer-Verlängerungsprodukt (P2.1) und das dritte Primer-Verlängerungsprodukt (P3.1) als Doppelstrang vorliegen;
   • - Hybridisieren eines vierten Primer-Oligonukleotids (P4.2) an das erste Primer-Verlängerungsprodukts (P1.1-Ext) und / oder an das dritte Primer-Verlängerungsprodukt (P3.1-Ext) wobei das vierte Primer-Oligonukleotid (P4.2) einen ersten Bereichs umfasst (P4.1.1), der sequenzspezifisch an den synthetisierten Bereich des ersten Primer-Verlängerungsprodukts (P1.1E1) und des dritten Primerverlängerungsproduktes (P3.1 E2) binden kann,
   • - Verlängerung des vierten Primer-Oligonukleotids (P4.2) durch die zweite Polymerase unter Erhalt eines vierten Primer-Verlängerungsproduktes (P4.2-Ext), welches neben dem vierten Primer-Oligonukleotid einen synthetisierten Bereich (P4.1E5 bis P4.1E2 oder P4.1E5 bis P4.1E1) umfasst, der im Wesentlichen komplementär zum ersten Primer-Verlängerungsprodukt (P1.1-Ext) oder identisch zur Zielsequenz ist, wobei das erste Primer-Verlängerungsprodukt (P1.1-Ext) und das vierte Primer-Verlängerungsprodukt (P4.1-Ext) als Doppelstrang vorliegen;
   • - Trennung des Doppelstrangs aus dem ersten Primer-Verlängerungsprodukt (P1.1-Ext) und dem vierten Primer-Verlängerungsprodukt (P4.2-Ext) und des Doppelstrangs aus dem zweiten Primer-Verlängerungsprodukt (P2.1-Ext) und dem dritten Primer-Verlängerungsprodukt (P3.1-Ext) und des Doppelstrangs aus dem vierten Primer-Verlängerungsprodukt (P4.1-Ext) und dem dritten Primer-Verlängerungsprodukt (P3.1-Ext);
   • - Hybridisieren des dritten Primer-Oligonukleotids (P3.2) an das vierte Primer-Verlängerungsprodukt (P4.2-Ext) und Verlängerung des dritten Primer-Oligonukleotids (P3.2) durch die zweite Polymerase; und
   • - Hybridisieren des vierten Primer-Oligonukleotids an das dritte Primer-Verlängerungsprodukt (P3.2-Ext) und Verlängerung des vierten Primer-Oligonukleotids (P4.2) durch die zweite Polymerase.

Method for amplifying a nucleic acid ( 60 ), comprising the steps:

1. a) a first amplification with the steps:
   • - Provision of a starting nucleic acid 1.1 which comprises a target sequence
   • Hybridizing a first primer oligonucleotide ( P1.1 ) to a nucleic acid to be amplified having a target sequence (either starting nucleic acid 1.1 or P2 .1-Ext), wherein the first primer oligonucleotide ( P1.1 ) comprises the following areas:
     • • a first area ( P1.1.1 ), the sequence-specific to a region of the nucleic acid to be amplified ( P2.1 E1 ),
     • • a second area ( P1.1.2 ) which is connected to the 5 'end of the first region or connected via a linker, which second region can be bound by a controller oligonucleotide and remains substantially uncoated for the polymerase used under the chosen reaction conditions;
   • Extension of the first primer oligonucleotide ( P1.1 ) by a first polymerase to give a first primer extension product ( P1.1 Text), which is next to the first primer oligonucleotide ( P1.1 ) comprises a synthesized region ( P1.1E1 to P1.1E4 ) which is substantially complementary to the nucleic acid or the target sequence to be amplified, wherein the first primer extension product and the nucleic acid to be amplified are present as a double strand;
   • - Binding of a Controller Oligonucleotide ( C1.2 ) to the first primer extension product ( P1.1 Text), the controller oligonucleotide ( C1.2 ) comprises the following areas:
     • • a first area ( C1.2.1 ) attached to the second region (overhang) of the first primer extension product ( P1.1E6 ),
     • • a second area ( C1.2.2 ) leading to the first region of the first primer oligonucleotide ( P1.1.1 ) is complementary, and
     • • a third area ( C1.2.3 ) comprising at least part of the synthesized region of the first primer extension product ( P1.1E4 ) is substantially complementary; and
     wherein the controller oligonucleotides ( C1.1 ) not as a template for a primer extension of the first primer oligonucleotide ( P1.1 ), and the first controller oligonucleotide ( C1.1 ) to the first primer extension product ( P1.1E6 . P1.1E5 . P1.1E4 ) while displacing the area complementary to this area ( P2.1 E1 . P2.1E2 ) binds to the nucleic acid to be amplified;
   • Hybridizing a second primer oligonucleotide ( P1.1 ) to the first primer extension product, wherein the second primer oligonucleotide ( P2 .1) comprises an area ( P2.1.1 ), sequence-specifically to the synthesized region of the first primer extension product ( P1.1E1 ),
   • Extension of the second primer oligonucleotide ( P.2.1 ) by the first polymerase to give a second primer extension product ( P2 .1-Ext) which is next to the second primer oligonucleotide ( P2.1 ) a synthesized area ( P2.1E4 to P2.1E1 ) which is substantially identical to the nucleic acid or the target sequence to be amplified, wherein the first primer extension product ( P1.1 -Ext) and the second primer extension product ( P2.1 ) form a first double-stranded amplification product; and
   • - Steps of the first amplification are repeated until the desired amount of first amplification products has been synthesized.
2. b) a second amplification with the steps:
   • Hybridizing a third primer oligonucleotide ( P3.2 ) to the second and / or the fourth primer extension product ( P1.1 -Ext. or P4.1 ), The third primer oligonucleotide ( P3.2 ) a first area ( P3.1 .1) which is sequence-specifically attached to a region of the second primer extension product ( P2.1E1 ) and the fourth primer extension product ( P4.1 E2 ),
   • Extension of the third primer oligonucleotide ( P3.2 ) by a second polymerase to give a third primer extension product ( P3.2 ) Which is next to the third primer oligonucleotide ( P3.2 ) a synthesized area ( P3.1 E5 to P3.1E2 respectively. P3.1E5 to P3.1E1 ) which is substantially complementary to the second primer extension product ( P2.1 -Text) or to the target sequence, wherein the second primer extension product ( P2.1 ) and the third primer extension product ( P3.1 ) are present as a double strand;
   • Hybridization of a fourth primer oligonucleotide ( P4.2 ) to the first primer extension product ( P1.1 Text) and / or to the third primer extension product ( P3.1 -Ext) where the fourth primer oligonucleotide ( P4.2 ) comprises a first area ( P4.1.1 ), sequence-specifically to the synthesized region of the first primer extension product ( P1.1E1 ) and the third primer extension product ( P3.1 E2 ),
   • Extension of the fourth primer oligonucleotide ( P4.2 ) by the second polymerase to give a fourth primer extension product ( P4.2 -Text), which besides the fourth primer oligonucleotide has a synthesized region ( P4.1E5 to P4.1E2 or P4.1E5 to P4.1E1 ) which is substantially complementary to the first primer extension product ( P1.1 ) Or identical to the target sequence, wherein the first primer extension product ( P1.1 -Ext) and the fourth primer extension product ( P4.1 -Ext) as a double strand;
   • Separation of the double strand from the first primer extension product ( P1.1 -Ext) and the fourth primer extension product ( P4.2 -Ext) and the double strand from the second primer extension product ( P2.1 -Ext) and the third primer extension product ( P3.1 -Ext) and the double strand from the fourth primer extension product ( P4.1 -Ext) and the third primer extension product ( P3.1 -Ext);
   • - Hybridizing the third primer oligonucleotide ( P3.2 ) to the fourth primer extension product ( P4.2 -Ext) and extension of the third primer oligonucleotide ( P3.2 ) by the second polymerase; and
   • Hybridizing the fourth primer oligonucleotide to the third primer extension product ( P3.2 -Ext) and extension of the fourth primer oligonucleotide ( P4.2 ) by the second polymerase.

Furthermore, the invention comprises the following aspects:

A method according to aspect 1, wherein the nucleic acid fragment comprising a first target sequence is provided in single-stranded form

The method of aspect 1, wherein the nucleic acid fragment comprising a first target sequence is provided in double stranded form (comprising a nucleic acid fragment including a first target sequence, the start nucleic acid chain, and a complementary nucleic acid fragment), and this double stranded nucleic acid fragment is converted to single stranded form prior to the first amplification becomes

The method of aspect 1, wherein the second amplification using the third and the fourth primer proceeds as a polymerase chain reaction (PCR).

Method according to aspect 1, in which the separation of the synthesized strands during the first amplification takes place predominantly sequence-specifically with the aid of controller oligonucleotide.

A method according to aspect 1, wherein the separation of the synthesized strands during the second amplification is effected under the action of a temperature which can cause separation of synthesized strands. For example, such a temperature includes ranges of 80 ° C to 105 ° C.

A method according to aspect 1, wherein the hybridization and primer extension during the second amplification is carried out at temperatures which allow a predominantly complementary binding of primers to complementary sequence segments of the synthesized strands and a primer extension by the second polymerase. For example, such temperature includes ranges from 20 ° C to about 80 ° C.

The method of aspect 1, wherein prior to the second amplification, separation of first and second primer extension products synthesized during the first amplification occurs so that both primer extension products are single-stranded.

The method of aspect 1, wherein the first primer oligonucleotide comprises in its first region a sequence segment which is capable of complementary binding to the first target sequence in its 3 'segment.

The method of aspect 1, wherein the second primer oligonucleotide comprises in its 3 'region a sequence segment which is substantially identical to a portion of the first target sequence, said portion being in the 5' segment of the target sequence

The method of aspect 1, wherein the controller oligonucleotide comprises in its second and third regions a sequence segment which is substantially identical to a portion of the target sequence.

The method of aspect 1, wherein the first target sequence and a strand complementary to this first target sequence are included on both sides of the primer pair comprising a first primer oligonucleotide and a second primer oligonucleotide.

The method of aspect 1, wherein the third primer oligonucleotide comprises in its 3 'region a sequence segment attached to the second primer extension product of the first amplification fragment 1.1 essentially complementary can bind.

The method of aspect 1, wherein the third primer oligonucleotide comprises in its 3 'region a sequence segment attached to the second primer extension product of the first amplification fragment 1.1 can substantially bind complementary and further comprises this 3 'region complementary segment to the first target sequence.

The method of aspect 1, wherein the fourth primer oligonucleotide comprises in its 3 'region a sequence segment attached to the first primer extension product of the first amplification fragment 1.1 essentially complementary can bind.

The method of aspect 1, wherein the fourth primer oligonucleotide comprises in its 3 'region a sequence segment attached to the first primer extension product of the first amplification fragment 1.1 essentially complementary to bind and further this 3 'region comprises a segment which is substantially identical to a portion of the first target sequence, wherein this proportion is in the 5' region of the target sequence

The method according to aspect 1, wherein the first target sequence is partially or completely and a strand complementary to this target sequence is included on both sides of the primer pair comprising a third primer oligonucleotide and a fourth primer oligonucleotide.

The method of aspect 1, wherein the first primer oligonucleotide and the third primer oligonucleotide are identical.

The method according to aspect 1, wherein the first primer oligonucleotide and the third primer oligonucleotide are identical, wherein during the second amplification resulting primer extension products ( P4.1- Ext part 1 ) and ( P4.1 -Ext) are identical.

The method of aspect 1, wherein the second primer oligonucleotide and the fourth primer oligonucleotide are identical.

The method according to aspect 1, wherein the second primer oligonucleotide and the fourth primer oligonucleotide are identical, wherein during the second amplification resulting primer extension products ( P3.1 -Ext part 1 ) and ( P3.1 -Ext) are identical.

The method of aspect 1, wherein the first primer oligonucleotide and the third primer oligonucleotide, and the second and the fourth primer oligonucleotide are identical.

A method according to aspect 1, wherein the first primer oligonucleotide and the third primer oligonucleotide, as well as the second and the fourth primer oligonucleotide are identical, wherein during the second amplification resulting primer extension products ( P4.1 -Ext part 1) and ( P4.1 -Ext) are identical and resulting primer extension products ( P3.1 -Ext part 1 ) and ( P3.1 -Ext) are identical.

Process by aspect 1 in which the second region of the first primer oligonucleotide comprises a polynucleotide tail which does not bind to the first target sequence.

The nucleic acid to be amplified in the first amplification comprises a first target sequence. Before the start of the first amplification, at least one start nucleic acid chain is generated 1.1 into the reaction which comprises a target sequence and can serve as a template for the synthesis of the first amplification fragment.

The synthesized primer extension products of the first amplification (the first amplification fragment 1.1 ) include the target sequence. This first amplification fragment 1.1 can be used as a start-up nucleic acid chain 2.1 serve with a template function for the second amplification.

The primer extension product synthesized in the second amplification ( P3.1 -Ext and P4.1 -Ext) (the second amplification fragment 2.1 ) comprise at least portions of the first target sequence.

The term "substantially complementary" in the sense of the invention means in particular that two nucleic acids have not more than 5, 4, 3, 2, or 1 mismatches to one another.

The term "substantially complementary" in the sense of the invention means in particular that the mutually complementary regions of nucleic acid have not more than 5, 4, 3, 2, or 1 mismatches.

In one embodiment of the method according to the invention, it is provided that the steps of the first amplification are repeated until the first amplification fragment ( 1.1 ) in a copy number in the range of 10 to 100,000,000,000, in particular from 100 to 1,000,000,000, in particular from 1,000 to 100,000,000.

In one embodiment of the method according to the invention, it is provided that the steps of the first amplification are repeated at least twice.

In a further embodiment of the method according to the invention, it is provided that the second amplification starting from a copy number of the first amplification fragments ( 1.1 ) is started, which is in the range of 10 to 100,000,000,000, in particular from 100 to 1,000,000,000, in particular from 1,000 to 100,000,000.

In a further embodiment of the method according to the invention, it is provided that the steps of the second amplification are repeated until the amplification fragment ( 2.1 ) in a number of copies in the range of 10,000 to 100,000,000,000,000, in particular from 100,000 to 100,000,000,000,000, especially from 1,000,000 to 100,000,000,000,000.

In one embodiment of the method according to the invention, it is provided that the steps of the second amplification are repeated at least twice.

In another embodiment, the detection system comprises at least one oligonucleotide probe labeled with a fluorescent reporter.

In another embodiment, the detection system comprises at least one oligonucleotide probe which is labeled with a fluorescent reporter and a fluorescence quencher.

In a further embodiment, the detection system comprises at least one oligonucleotide probe labeled with a donor fluorophore capable of forming a FRET pair with the fluorescent reporter.

In another embodiment, an oligonucleotide probe comprises at least one of the formed primer extension products ( P1.1 -ext, P2.1 -ext, P3.1 -ext, P4.1 -Ext) substantially complementary sequence segment which can hybridize to at least one of the primer extension product formed under suitable conditions (hybridization conditions).

In a further embodiment, this sequence segment is complementary to the first and / or the third primer extension product.

In a further embodiment, this sequence segment is complementary to the first and / or the third primer extension product, wherein the oligonucleotide probe can complementarily bind in the 3 'segment of the respective primer extension product which is not complementarily bound by the controller oligonucleotide.

In a further embodiment, this sequence segment is complementary to the second and / or the fourth primer extension product.

In a further embodiment, this sequence segment of the oligonucleotide probe comprises a length which is in the range between 10 nucleotides and 50, more preferably between 15 and 40, preferably between 15 and 30 nucleotides.

In another embodiment, this sequence segment of the oligonucleotide probe does not comprise a sequence region that is substantially complementary to the controller oligonucleotide.

In another embodiment, this sequence segment of the oligonucleotide probe comprises a sequence region which is substantially complementary to the controller oligonucleotide, the length of this segment being less than 20 nucleotides, better still less than 15 nucleotides, preferably less than 10 nucleotides,

In another embodiment, this sequence segment of the oligonucleotide probe does not comprise a sequence region that is substantially complementary to one of the primer oligonucleotides.

In a further embodiment, this sequence segment of the oligonucleotide probe comprises a sequence region that is substantially complementary to one of the primer oligonucleotides, the length of this segment being less than 20 nucleotides, better less than 15 nucleotides, preferably less than 10 nucleotides, more preferably less than 5 nucleotides.

In another embodiment, this sequence segment of the oligonucleotide probe does not include a sequence region that is substantially identical to the sequence of the third region of the controller oligonucleotide.

In another embodiment, this sequence segment of the oligonucleotide probe comprises a sequence region which is substantially identical to the sequence of the third region of the controller oligonucleotide, the length of this segment being less than 20 nucleotides, more preferably less than 15 nucleotides, preferably less than Includes 10 nucleotides,

In one embodiment, the controller oligonucleotide comprises one of the following components (a fluorescence reporter and / or a fluorescence quencher and / or a donor fluorophore) with at least one of these components located in the third region of the controller oligonucleotide.

In one embodiment, the oligonucleotide probe is at least partially cleavable by 5'-3'-nuclease.

In another embodiment, an oligonucleotide probe comprises a sequence segment which is substantially complementary to the target sequence or its portion encompassed by one of the amplification products, the length of this segment being in the range of 5 to 50 nucleotides, more preferably 10 to 10 nucleotides 40, preferably between 15 and 30 nucleotides.

In another embodiment, an oligonucleotide probe does not comprise a sequence segment substantially complementary to the target sequence or its complementary strand.

The binding of the oligonucleotide probe to the respective complementary segment of the primer extension product formed under suitable hybridization conditions leads to formation of a double strand. The hybridization conditions are present during the process.

At appropriate times during the process, the reaction is illuminated with light of a suitable wavelength for generation of a fluorescence signal and detection of the fluorescence signal from the fluorescence reporter, whereby the intensity of the fluorescence signal is measured.

The detection of the fluorescence signal is carried out with suitable optical / physical means, in which either the increase of the fluorescence signal or the decrease of the fluorescence signal is measured. By means of suitable sensors, the intensity of the fluorescence signal can be converted into measured values which, after appropriate calibration, make it possible to determine whether a desired target sequence is present in the reaction mixture or not.

An essential aspect of the fluorescence detection system is the reporter-quencher pair. When the quencher is in close proximity to the reporter, no signal is emitted upon exposure to the excitation light. When reporter and quencher molecules are held in close proximity due to a complementary stem sequence, no fluorescence signal appears even under illumination. However, if the fluorescence system is altered such that the nucleotide sequence located between reporter and quencher can hybridize to a target sequence, then reporters and quenchers are placed in a spatial distance that causes the quencher to be so far from the reporter molecule that the reporter molecule will emits fluorescence radiation when the reaction mixture is irradiated with an excitation light source. The distance between reporter and quencher depends on the molecules used. Usually, the emission of light after irradiation of the fluorescence reporter is then reduced or almost completely reduced when the quencher and reporter are at a distance of less than 25 nucleotides from each other. This removal can be effected either by the nucleotide sequence or by special spacing molecules, such as linkers or spacers.

In another embodiment, the fluorescent reporter can form a specific reporter-donor (FRET) pair with a donor fluorophore capable of transmitting the absorbed energy to fluorescent reporters by fluorescence resonance energy transfer (FRET) ,

A reporter-donor pair comprising a fluorescent reporter and a suitable donor fluorophore and forming a fluorescence resonance energy transfer pair may be such that only one of the partners of such a FRET pair is attached to the oligonucleotide probe is coupled and the other partner is coupled to the controller oligonucleotide. Both fluorescent reporter and donor fluorophore are coupled to the respective oligonucleotide such that in the non-hybridizing state of the oligonucleotide probe, the donor fluorophore is unable to transfer the absorbed energy to the fluorescent reporter.

In a preferred embodiment, the controller oligonucleotide is a component of the detection system and comprises a fluorescent reporter and / or a fluorescence quencher and / or a donor fluorophore. Simultaneous hybridization of the controller oligonucleotide to the synthesized first or third primer extension product and the oligonucleotide probe to the 3 'segment of the same first and / or third primer extension product binds to adjacent sequence positions of the first and / or third primer Extension product, resulting in spatial proximity of the donor fluorophore and the fluorescence reporter, and the distance between the fluorescence reporter and donor fluorophore is reduced so much that FRET can proceed from donor fluorophore to fluorescent reporter. This leads to the generation of a fluorescence signal of the fluorescence reporter and results in a detectable increase in the fluorescence signal of the fluorescence reporter.

• • Die Kopplung am Controller -Oligonukleotid erfolgt bevorzugt im dritten Bereich des Aktivator-Oligonukleotides,
• • Die Kopplung am Controller -Oligonukleotid erfolgt bevorzugt am 5'-Ende oder des dritten Bereichs oder in seiner Nähe, z.B. 2 bis etwa 10 Nukleotide vom 5'-Ende des dritten Bereichs
• • Kopplung an der Oligonukleotid-Sonde erfolgt bevorzugt im mittleren Bereich der Oligonukleotid-Sonde,
• • Kopplung an der Oligonukleotid-Sonde erfolgt bevorzugt im 3'-Segment der Oligonukleotid-Sonde.

The distance between the donor fluorophore and the fluorescent reporter after hybridization should be less than 25, preferably less than 15, and more preferably less than 5 nucleotides. The wavelength of the light upon excitation is absorbed by the donor fluorophore and transferred to the reporter, thereby emitting light that can be detected. As regards the arrangement of the detection system in the amplification product, the following embodiments are preferred, wherein either the fluorescence reporter or the fluorescence quencher or the donor fluorophore are coupled either to the controller oligonucleotide, in particular in the third region of the controller oligonucleotide:

• The coupling to the controller oligonucleotide preferably takes place in the third region of the activator oligonucleotide,
• The coupling to the controller oligonucleotide is preferably at the 5 'end or the third region or in its vicinity, for example 2 to about 10 nucleotides from the 5' end of the third region
• Coupling to the oligonucleotide probe is preferably carried out in the middle region of the oligonucleotide probe,
• • Coupling to the oligonucleotide probe is preferably carried out in the 3 'segment of the oligonucleotide probe.

• • Der Abstand zwischen beiden Elementen eines FRET-Paares im hybridisierten Zustand von Oligonukleotiden, welche an das erste Primer-Verlängerungsprodukt hybridisiert sind, beträgt weniger als etwa 30 Nukleotide.
• • Die Oligonukleotid-Sonde, die ein komplementäres 3'-Segment zu einem der Primer-Verlängerungprodukte umfasst, ist dermaßen modifiziert, dass die Polymerase nicht in der Lage ist, dieses 3'-Ende zu verlängern.
• • Die Oligonukleotid-Sonde, die ein komplementäres 3'-Segment zu einem der Primer-Verlängerungprodukte umfasst, dass die Polymerase in der Lage ist, dieses 3'-Ende zu verlängern, wobei die Oligonukleotid-Sonde als ein PCR-Primer dient.
• • Verfahren zur Detektion von Amplifikation, wobei zwei oder mehr Nukleinsäureketten amplifiziert werden, bei welchem für jede zu amplifizierende Nukleinsäurekette ein spezifisches Detektions-System verwendet wird.
• • Verfahren zur Detektion von Amplifikation, wobei zwei oder mehr zu amplifizierende Nukleinsäureketten amplifiziert werden, bei welchem die Detektion der Amplifikation von mindestens zwei zu amplifizierenden Nukleinsäureketten durch ein einheitliches Detektions-System erfolgt.
• • Die Amplifikation umfasst eine asymetrische Vermehrung eines der Primer-Verlängerungsprodukte.
• • Die Anregung des Fluoreszenzreporters und die Messung des Fluoreszenzsignals des Fluoreszenzreporters erfolgt während der Amplifikation.
• • Die Anregung des Donor-Fluorophores und die Messung des Fluoreszenzsignals des Fluoreszenzreporters erfolgt während der Amplifikation.
• • Die Anregung des Fluoreszenzreporters und die Messung des Fluoreszenzsignals des Fluoreszenzreporters erfolgt nach der Amplifikation.
• • Die Anregung des Donor-Fluorophores und die Messung des Fluoreszenzsignals des Fluoreszenzreporters erfolgt nach der Amplifikation.
• • Die Oligonukleotid-Sonde ist ein DNA-Oligonukleotid.
• • Die Oligonukleotid-Sonde ist ein DNA-Oligonukleotid und das 3'-Ende blockiert ist, so dass Polymerase es nicht verlängern kann.
• • Die Oligonukleotid-Sonde umfasst ein komplementäres Sequenz-Segment zum ersten und / oder dem dritten Primer-Verlängerungsprodukt, wobei dieses Sequenz-Segment eine Länge von 8 bis etwa 60 Nukleotide umfasst.
• • Eine Oligonukleotid-Sonde umfasst ein komplementäres Sequenz-Segment zum 3'-Segment des ersten und / oder des dritten Primer-Verlängerungsprodukt, welches nicht vom Controller-Oligonukleotid gebunden wird, wobei dieses Segment eine Länge von 8 bis etwa 40 Nukleotide umfasst.

In the context of the present invention, the following aspects are preferred:

• The distance between both elements of a hybridized FRET pair of oligonucleotides hybridized to the first primer extension product is less than about 30 nucleotides.
• The oligonucleotide probe comprising a complementary 3 'segment to one of the primer extension products is modified so that the polymerase is unable to extend that 3' end.
• The oligonucleotide probe comprising a complementary 3 'segment to one of the primer extension products such that the polymerase is capable of extending that 3' end, the oligonucleotide probe serving as a PCR primer.
• A method for detecting amplification, wherein two or more nucleic acid chains are amplified in which a specific detection system is used for each nucleic acid chain to be amplified.
• A method for detecting amplification, wherein two or more nucleic acid chains to be amplified are amplified, wherein the detection of the amplification of at least two nucleic acid chains to be amplified by a uniform detection system.
• • The amplification involves an asymmetric amplification of one of the primer extension products.
• The excitation of the fluorescence reporter and the measurement of the fluorescence signal of the fluorescence reporter takes place during the amplification.
• The excitation of the donor fluorophore and the measurement of the fluorescence signal of the fluorescence reporter occur during the amplification.
• The excitation of the fluorescence reporter and the measurement of the fluorescence signal of the fluorescence reporter takes place after the amplification.
• The excitation of the donor fluorophore and the measurement of the fluorescence signal of the fluorescent reporter takes place after the amplification.
• The oligonucleotide probe is a DNA oligonucleotide.
• The oligonucleotide probe is a DNA oligonucleotide and the 3 'end is blocked so that polymerase can not extend it.
• The oligonucleotide probe comprises a complementary sequence segment to the first and / or the third primer extension product, which sequence segment is from 8 to about 60 nucleotides in length.
• An oligonucleotide probe comprises a complementary sequence segment to the 3 'segment of the first and / or third primer extension product which is not bound by the controller oligonucleotide, which segment is from 8 to about 40 nucleotides in length.

An oligonucleotide probe comprises at least one further modification selected from the following group: linkers (eg HEG, C3, C6), phosphate-sugar-backbone modifications (eg PTO, 2'-O-Me, RNA, PNA, LNA modifications) ,

In a further embodiment of the method according to the invention, it is provided that the third primer oligonucleotide and / or the fourth primer oligonucleotide have a second region which adjoins the 5 'end of the first region or is linked via a linker, wherein the second region can not bind complementary to the first primer extension product or to the second primer extension product.

In a further embodiment of the method according to the invention, it is provided that the second region of the third and / or fourth primer oligonucleotide lies in the 5 'direction from the first region of the third and / or fourth primer oligonucleotide.

In a further embodiment of the method according to the invention, it is provided that the third primer oligonucleotide and / or the fourth primer oligonucleotide comprises a barcode sequence.

In a further embodiment of the method according to the invention, it is provided that the separation of the double strand from the first primer extension product and the fourth primer extension product and the double strand from the second primer extension product and the third primer extension product is carried out by thermal denaturation, in particular at a temperature in the range of 85 ° C to 105 ° C.

In a further embodiment of the method according to the invention it is provided that the first polymerase is capable of strand displacement during the synthesis.

In a further embodiment of the method according to the invention, it is provided that the second polymerase is a thermostable polymerase.

In a further embodiment of the method according to the invention, it is provided that the first polymerase and the second polymerase are identical.

In a further embodiment of the method according to the invention, it is provided that the first primer oligonucleotide and the third primer oligonucleotide are substantially identical.

In a further embodiment of the method according to the invention, it is provided that the second primer oligonucleotide ( P2.1 ) and the third primer oligonucleotide ( P4.2 ) are substantially identical.

The term "substantially identical" in the context of the invention means in particular that two nucleic acid sequences with not more than 5, 4, 3, 2, or 1 Mismatches to each other.

In a further embodiment of the method according to the invention, it is provided that the reaction conditions of the first amplification are selected so that spontaneous dissociation of the first amplification product can not occur.

In a further embodiment of the method according to the invention, it is provided that the second amplification is carried out at at least two temperatures, wherein at a first temperature the hybridization and extension of the third and / or fourth primer oligonucleotide to the first and / or second and / or third and / or fourth primer extension product, and at a second temperature separation of the resulting duplex.

In a further embodiment of the method according to the invention, it is provided that the second amplification is carried out at at least three temperatures, wherein at a first temperature the hybridization of the third and / or fourth primer oligonucleotide to the first and / or second and / or third and / or or fourth primer extension product takes place, at a second temperature the extension of the third and / or fourth primer, and at a third temperature the separation of the resulting double strands.

In a further embodiment of the method according to the invention, it is provided that the first amplification and the second amplification are carried out in the same reaction mixture.

In a further embodiment of the method according to the invention, it is provided that the second polymerase, the third primer oligonucleotide and / or the fourth primer oligonucleotide can be activated, and / or the controller oligonucleotide can be deactivated.

Activatable polymerases may be, for example, reversible inactivated polymerases such as thermostable polymerase (hot-start polymerase) or polymerases which are reversibly inactivated by an antibody or a chemical modification.

Activatable primers may be reversible inactivated primers, for example oligonucleotides with protected 3'-OH group, the protecting group preferably being removable by a polymerase having 5 'exonuclease activity. It would be advantageous in the first amplification to use a polymerase without 5'-exonuclease activity.

The controller oligonucleotide can be inactivated or cleaved, for example, by a polymerase with 5 'exonuclease activity. Alternatively, the controller oligonucleotide may also be cleaved and thereby removed by other enzymatic or chemical methods prior to the second amplification.

In one embodiment of the method according to the invention, it is provided that the first polymerase is inactivated before carrying out the second amplification. This can be achieved, for example, by thermal denaturation, when the first polymerase is a non-thermostable polymerase.

In a further embodiment of the method according to the invention, it is provided that the first amplification is carried out in a first reaction mixture and the second amplification in a second reaction mixture.

In a further embodiment of the method according to the invention, it is provided that an aliquot of the first reaction mixture is added to the second reaction mixture.

In a further embodiment of the method according to the invention, it is provided that the complete first reaction mixture is added to the second reaction mixture.

In one embodiment of the method according to the invention, it is provided that the third region of the controller oligonucleotide is substantially complementary to the part of the synthesized region of the first primer extension product which directly adjoins the primer oligonucleotide portion of the first primer extension product. Advantageously, this improves the sequence-specific displacement of the nucleic acid to be amplified.

In a further embodiment of the method according to the invention, it is provided that the controller oligonucleotide comprises a fourth region which can bind in a substantially sequence-specific manner to the 3 'region of the third primer oligonucleotide. The fourth region may be within the second region and / or the third region of the controller oligonucleotide, or include second and / or third region sequence segments, wherein the fourth region preferably has a length of from 9 to 30 nucleotides, more preferably 10 to 20 Nucleotides, in particular 12 to 16 nucleotides.

The term "essentially sequence-specific" in the context of the invention means in particular that the mutually complementary regions of the primer oligonucleotide and the nucleic acid to be amplified have not more than 5, 4, 3, 2, or 1 mismatches.

In a further embodiment of the invention it is provided that the controller oligonucleotide comprises one or more nucleotide modifications, which are designed to prevent the extension of a primer bound to the controller oligonucleotide. For example, multiple 2'-O-alkyl modifications may block or prevent such extension. Preferably, there are several nucleotide modifications in the second and / or third region of the controller oligonucleotide. Preferably, the controller oligonucleotide comprises at least 5 such modifications, more preferably at least 10 modifications.

In a preferred embodiment, the controller oligonucleotide consists entirely of nucleoid modifications.

In another embodiment of the method of the invention, the portion of the polymerase-synthesized portion of the first primer extension product that is substantially complementary to the third portion of the controller nucleotide has a length in the range of 5 nucleotides to 70 nucleotides.

In one embodiment, the third single-stranded region of the controller oligonucleotide is fully complementary to said 5 'segment of the first primer extension product, the length of this complementary sequence segment comprising: at least 3 to 70 nucleotides, more preferably at least 5 to 50 nucleotides , preferably from 5 to 40 nucleotides, more preferably from 5 to 30 nucleotides, more preferably from 5 to 20 nucleotides.

In a further embodiment of the method according to the invention, it is provided that the first amplification is carried out essentially isothermally.

In a further embodiment of the method according to the invention it is provided that the second amplification is carried out at three temperatures, wherein at the first temperature, the hybridization of the third or fourth primer oligonucleotide and optionally the initial extension of the primer is performed, and at a second temperature complete extension of the primers and at a third temperature the separation of formed primer extension products from their respective template strands. Preferably, the first temperature is in the range of 25 ° C to 65 ° C and the second temperature in the range of 65 ° C to 80 ° C and the third temperature in the range of 85 ° C to 105 ° C.

In a further embodiment of the method according to the invention, it is provided that the nucleic acid to be amplified has a length in the range from 20 nucleotides to 300 nucleotides.

In a further embodiment of the method according to the invention, it is provided that the first primer oligonucleotide has a length in the range from 15 nucleotides to 100 nucleotides.

In a further embodiment of the method according to the invention, it is provided that the second primer oligonucleotide has a length in the range from 15 nucleotides to 100 nucleotides.

In a further embodiment of the method according to the invention, it is provided that the controller oligonucleotide has a length in the range from 20 nucleotides to 100 nucleotides.

In a further embodiment of the method according to the invention, it is provided that the third primer oligonucleotide has a length in the range from 15 nucleotides to 60 nucleotides.

In a further embodiment of the method according to the invention, it is provided that the fourth primer oligonucleotide has a length in the range from 15 nucleotides to 60 nucleotides.

In a further embodiment of the method according to the invention, it is provided that the third primer oligonucleotide has at least one nuclease-resistant nucleotide modification, for example a PTO, or 2'-O-alkyl modification.

In a further embodiment of the method according to the invention, it is provided that the fourth primer oligonucleotide has at least one nuclease-resistant nucleotide modification, for example a PTO, or 2'-O-alkyl modification.

In a further embodiment of the method according to the invention, it is provided that the first primer oligonucleotide has a modification or several modifications in the second region, in particular immediately following the first region of the first primer oligonucleotide, which stops the polymerase used at the copying second region. prevented. Advantageously, only the first region of the first primer oligonucleotide is thereby copied.

• - ein erstes Primer-Oligonukleotid aufweisend folgende Bereiche:
  • • einen ersten Bereich, der sequenzspezifischen an einen Bereich der zu amplifizierenden Nukleinsäure binden kann,
  • • einen zweiten Bereich, der sich an das 5'-Ende des ersten Bereichs anschließt oder über einen Linker verbunden ist, wobei der zweite Bereich von einem ersten Controller-Oligonukleotids gebunden werden kann und von einer für die Amplifikation verwendete Polymerase unter den gewählten Reaktionsbedingungen im Wesentlich unkopiert bleibt;
  • • wobei das erste Primer-Oligonukleotid durch eine Polymerase zu einem ersten Primer-Verlängerungsprodukt verlängert werden kann, welches neben dem ersten Primer-Oligonukleotid einen synthetisierten Bereich umfasst;
• - ein zweites Primer-Oligonukleotid, wobei das zweite Primer-Oligonukleotid einen Bereich umfasst, der sequenzspezifisch an den synthetisierten Bereich des ersten Primer-Verlängerungsprodukts binden kann, und von einer Polymerase zu einem zweiten Primer-Verlängerungsprodukt verlängert werden kann, welches neben dem zweiten Primer-Oligonukleotid einen synthetisierten Bereich umfasst
• - ein Controller-Oligonukleotids aufweisend folgende Bereiche:
  • • einen ersten Bereich, der an den zweiten Bereich des ersten Primer-Verlängerungsprodukts binden kann,
  • • einen zweiten Bereich, der zum ersten Bereich des ersten Primer-Oligonukleotid im Wesentlichen komplementär oder vollständig ist, und
  • • einen dritten Bereich, der zumindest zu einen Teil des synthetisierten Bereiches des ersten Primer-Verlängerungsprodukts im Wesentlichen komplementär oder vollkomplementär ist; wobei das Controller-Oligonukleotid nicht als Matrize für eine Primerverlängerung des ersten Primer-Oligonukleotids dient, und das Controller-Oligonukleotid an das Primer-Verlängerungsprodukt unter Verdrängung des dazu komplementären Bereichs der zweiten Primer-Verlängerungsproduktes binden kann
• - ein drittes Primer-Oligonukleotid, wobei das dritte Primer-Oligonukleotid einen ersten Bereichs umfasst, der sequenzspezifisch an ein Segment des zweiten Primer-Verlängerungsprodukt binden kann, und von eine Polymerase zu einem dritten Primer-Verlängerungsprodukt (P3.1-Ext) verlängert werden kann, welches neben dem dritten Primer-Oligonukleotid einen synthetisierten Bereich umfasst; und
• - ein viertes Primer-Oligonukleotid, wobei das vierte Primer-Oligonukleotid einen ersten Bereichs umfasst, der sequenzspezifisch an den synthetisierten Bereich des ersten Primer-Verlängerungsprodukts binden kann, und von einer Polymerase zu einem vierten Primer-Verlängerungsprodukt verlängert werden kann, welches neben dem vierten Primer-Oligonukleotid einen synthetisierten Bereich umfasst.

According to a further aspect of the invention, a kit is provided for carrying out the inventive method according to the first aspect or one of the associated embodiments or alternatives. The kit according to the invention comprises:

• a first primer oligonucleotide having the following ranges:
  • A first region which can bind sequence-specific to a region of the nucleic acid to be amplified,
  • A second region adjoining the 5 'end of the first region or connected via a linker, which second region can be bound by a first controller oligonucleotide and by a polymerase used for the amplification under the selected reaction conditions in Essentially remains uncopied;
  • Wherein the first primer oligonucleotide can be extended by a polymerase to a first primer extension product comprising a synthesized region in addition to the first primer oligonucleotide;
• a second primer oligonucleotide, wherein the second primer oligonucleotide comprises a region that can sequence-specifically bind to the synthesized region of the first primer extension product and can be extended from a polymerase to a second primer extension product that is adjacent to the second primer Oligonucleotide comprises a synthesized region
• a controller oligonucleotide comprising the following ranges:
  • A first region that can bind to the second region of the first primer extension product,
  • A second region which is substantially complementary or complete to the first region of the first primer oligonucleotide, and
  • A third region that is substantially complementary or fully complementary to at least a portion of the synthesized region of the first primer extension product; wherein the controller oligonucleotide does not serve as a template for primer extension of the first primer oligonucleotide and can bind the controller oligonucleotide to the primer extension product displacing the complementary region of the second primer extension product
• a third primer oligonucleotide, wherein the third primer oligonucleotide comprises a first region capable of sequence-specific binding to a segment of the second primer extension product, and a polymerase to a third primer extension product ( P3.1 -Ext) comprising a synthesized region in addition to the third primer oligonucleotide; and
• a fourth primer oligonucleotide, wherein the fourth primer oligonucleotide comprises a first region capable of sequence-specific binding to the synthesized region of the first primer extension product and can be extended from a polymerase to a fourth primer extension product which is adjacent to the fourth Primer oligonucleotide comprises a synthesized region.

In one embodiment of the kit according to the invention, the kit further comprises at least one polymerase.

In one embodiment of the kit according to the invention, the kit comprises a first polymerase intended for extension of the first and second primer oligonucleotide and a second polymerase intended for extension of the third and the fourth primer oligonucleotide.

In one embodiment of the kit according to the invention, the first polymerase has no 5 'exonuclease activity and the second polymerase no 5' exonuclease activity. Preferably, the second polymerase is a thermostable polymerase.

In one embodiment of the kit according to the invention, it is provided that the second polymerase, the third primer oligonucleotide and / or the fourth primer oligonucleotide can be activated, and / or the controller oligonucleotide can be deactivated.

According to a further aspect of the invention, the use of a kit according to the above aspect for performing the inventive according to the first aspect or one of the associated embodiments or alternatives is provided.

Further details and advantages of the invention will be explained in a non-restrictive manner by the following figures and a detailed description of selected embodiments.

list of figures

• 1 schematically shows the components of an embodiment of the amplification method according to the invention in a heterogeneous format; A) of the first amplification system; B) of the second amplification system.
• 2 schematically shows the components of an embodiment of the first amplification system;
• 3 shows a temperature profile of an embodiment of the method according to the invention in the heterogeneous format;
• 4 shows a temperature profile of another embodiment of the method according to the invention in the heterogeneous format;
• 5 shows the components of an embodiment of the amplification method according to the invention in a homgenen format; A) of the first amplification system; B) of the second amplification system.
• 6 to 11 shows temperature profiles of embodiments of the method according to the invention;
• 12 schematically shows the structure of a first primer oligonucleotide;
• 13 schematically shows the complementary binding of the first primer to the nucleic acid chain to be amplified;
• 14 shows schematically the complementary binding of the first primer to the nucleic acid chain to be amplified and the extension of the primer;
• 15 schematically shows a primer extension product of the first primer;
• 16 schematically shows the relation between the individual components during the synthesis of the first primer extension product;
• 17 schematically shows the synthesis of the first primer extension product;
• 18 schematically shows the synthesis of the second primer extension product;
• 19 schematically shows the synthesis of the second primer extension product;
• 20 schematically shows the synthesis of the third and fourth primer extension product;
• 21 schematically shows the synthesis of the partial third primer extension product;
• 22 schematically shows the synthesis of the partial third primer extension product;
• 23 schematically shows the synthesis of the complete fourth primer extension product;
• 24 schematically shows the synthesis of the complete fourth primer extension product;
• 25 schematically shows the synthesis of the complete third primer extension product;
• 26 schematically shows the synthesis of the complete third primer extension product;
• 27 to 34 schematically show the relations between the individual components during the synthesis of the first primer extension product and the third PCR primer;
• 35 to 39 schematically show the components of an embodiment of the first amplification system;
• 40 schematically shows a primer oligonucleotide (A) and a nucleic acid to be amplified (B);
• 41 schematically shows strand displacement by an activator oligonucleotide;
• 42 schematically shows the interactions between in a reaction of the method according to the invention;
• 43 shows schematically an amplification by simultaneous synthesis of two strands;
• 44 to 56 show the results of example experiments.
• 57-58 shows schematically a preparation of a starting nucleic acid chain starting from genomic DNA.

Detailed description of the invention

In particular, the above-mentioned objects of the present invention are achieved by combining two consecutive amplifications, PCR amplification being the final step.

The combination according to the invention comprises two amplification processes to be carried out in succession. During the first partial amplification (the first amplification procedure), nucleic acid chains comprising a target sequence are amplified. This results in amplified nucleic acid chains, which are then used as templates in the second partial amplification (the second amplification method). This second partial amplification is preferably a conventional PCR. During the second partial amplification, the amplification proceeds from the target nucleic acids or nucleic acids comprising the target nucleic acids which were amplified in the first partial amplification. In this case, PCR fragments are amplified. These PCR fragments preferably comprise target sequences or portions of target sequences. Furthermore, during the second amplification process, specific changes can be made to amplifiable nucleic acid chains. For example, target sequences can be flanked by using barcoding primers or detection-specific primers. Also, detection can be detected using PCR probes (e.g., so-called Taqman probes). Furthermore, immobilization of target sequences to the solid phase using immobilized primers can be used during the PCR phase, so that a PCR proceeds as a solid-phase PCR.

The first amplification method according to the invention is intended to be able to synthesize or amplify nucleic acid chains having a defined sequence composition, it being possible for generated products to serve as start nucleic acid chains with template function for subsequent PCR and for the PCR amplification to be carried out using at least one own PCR primer ,

An object of the invention is further achieved by providing a first amplification method (a first partial amplification) and corresponding means for carrying it out. The execution of this amplification process was described in the PCT application PCT / EP2017 / 071011 and the European application 16185624.0 already described. For details of the embodiment of the amplification, the skilled person is referred to this application. This application is hereby incorporated in full (incorporated by reference)

The second amplification method (the second partial amplification) is preferably carried out as PCR, wherein at least one of the primers used has a different composition and / or structure than the primers used in the first amplification method. This results in an increase of at least one nucleic acid chain comprising specific target sequence or its parts.

As such, PCR amplification per se is normally capable of generating well-characterized amplification products from the initial template, more specifically in real-time mode, starting from an amount greater than about 1000 copies, more preferably greater than about 100,000 copies Generate signals. Reducing the number of copies to less than 1000 (eg, 100 or 10 copies) requires more cycles of PCR synthesis, which can progressively lead to defective amplification products or signals. This is especially the case if the PCR has to amplify a sequence variant (eg a mutation or allelic variant of a sequence) which is present in small amounts in the batch and the PCR amplification in the presence of a large amount of another sequence variant (eg. Sequence or Allel variant) takes place. Although a variety of methods have been developed to improve the specificity of PCR, amplification-dependent detection methods can benefit from further enhancement of the specificity of amplification techniques, eg in the field of liquid biopsy. Especially in clinical diagnostics, there is a sustained need to improve the specificity of measurement methods.

Due to the widespread use of PCR technology, there are numerous modifications of this technology or extensions of the PCR base process, which find application in many fields of molecular biology. By pre-connecting another, first amplification method, for example, signal specificity or PCR product specificity can be improved in these applications.

In the present invention, the combination of a preceding highly specific amplification method with downstream PCR will be described. The combination makes it possible to increase the initial copy number of nucleic acid chains to be amplified under highly selective amplification conditions of a first amplification method up to the desired amounts, which are then subsequently further amplified under less specific conditions of a PCR amplification. The first amplification method (first partial amplification) thus has a function of the first amplifier with high specificity. For example, the amount of synthesized copies in the first amplification process will range from about 10 copies to about 100,000,000,000 copies, more preferably from about 100 copies to about 1,000,000,000 copies, preferably from about 1,000 to about 100,000,000 copies. In this case, the amount of a target sequence is increased by the first amplification. This increase (based on the initial amount of the target sequence in the batch) is, for example, in the following ranges from about 2 times to about 10,000,000,000 times, better from about 10 times to about 1,000,000,000 times, even better from about 10 times. to about 1,00,0000,000 times, preferably from about 10 times to about 10,000,000 times, more preferably from about 100 times to 1,000,000 times. In this case, based on the volume of the reaction, the following concentration ranges are achieved: from about 10 amol / l to about 10 nmol / l, more preferably from about 1 fmol / l to about 1 nmol / l, even better from about 10 fmol / l to about 0.1 nmol / l, preferably from about 10 fmol / l to about 10 pmol / l.

The second amplification method (the second partial amplification, which is used as PCR method or one of its modifications) plays the role of a second amplifier and allows existing PCR-based methods, such as probe insertion or bar coding or sequence coding in NGS library preparation or solid-phase PCR in combination with specific products of the first amplification.

The advantages of the combination are on the one hand in reducing the necessary number of PCR cycles, which are necessary for the execution of the amplification up to desired amounts of the final product. This reduces the likelihood or extent of synthesis of products by PCR. The synthesis result can thus have higher specificity than the PCR amplification alone.

In a preferred embodiment, first the first amplification with required components of the first amplification system occurs in the absence of at least one of the essential components of the second amplification system in the same reaction mixture during the first amplification reaction. For example, PCR primers or a thermostable polymerase are added only prior to PCR amplification.

After completion of the first amplification reaction, the reaction mixture is brought into contact with the components of the second amplification system and PCR amplification is carried out.

In a preferred embodiment, the reaction components of a respective amplification system are present before the beginning of an amplification in separate reaction mixtures and / or in separate reaction vessels.

For example, the first amplification reaction is carried out in one reaction vessel, and then the synthesis result of this reaction is transferred (completely or partially) to the second reaction vessel, and then the second amplification is carried out.

In a further preferred embodiment, the reaction components are added sequentially, so that first the components of the first amplification system are added, and the first amplification reaction is carried out. Subsequently, the components of the second amplification system are supplied, so that the second amplification can take place. In a further preferred embodiment, the addition of the components of the second amplification system to the first takes place Amplification approach, so that the reaction is carried out substantially in the same reaction vessel. The sequence of both amplification methods is thus predetermined by the time sequence of the addition of individual components.

To reduce the likelihood of contamination, both reactions may preferably be carried out in a closed system. Such a system may comprise, for example, at least two separate reaction vessels. The transfer of the batch from one reaction vessel to the other preferably also takes place by means of a closed system. Such a separation of the two reactions in a closed reaction vessel system is known. Many arrangements of such separate reaction vessels are known which are grouped together to form a cartridge system or array system or microfluidic system.

In a preferred embodiment, the use of the first amplification mixture comprising the amplified nucleic acids of the first amplification for the second amplification without division of the first approach. The first approach is thus essentially completely fed to the second amplification reaction. This can be done, for example, by adding or adding the components of the second amplification system to the first reaction mixture. An amount of synthesized copies from the first amplification process is supplied thereto to the second amplification system and, for example, ranges from about 10 copies to about 100,000,000,000 copies, more preferably from about 100 copies to about 1,000,000,000 copies, preferably from about 1,000 to about 100,000 .000 copies. In this case, the amount of a target sequence is increased by the first amplification. This increase (based on the starting amount of the target sequence in the approach) is, for example, even better in the following ranges from about 2 times to about 10 000 000 000 times, better still from about 10 times to about 1 000 000 000 times from about 10 times to about 1,00,0000,000 times, preferably from about 10 times to about 10,000,000 times, more preferably from about 100 times to 1,000,000 times. Based on the volume of the reaction, the following concentration ranges can be achieved: from about 10 amol / l to about 10 nmol / l, more preferably from about 1 fmol / l to about 1 nmol / l, even better from about 10 fmol / l to about 0 , 1 nmol / l, preferably from about 10 fmol / l to about 10 pmol / l.

In a further preferred embodiment, it is preferred to use only a portion of the first amplification mixture (an aliquot or a subset) in the second amplification reaction. Due to an amplification by means of the first amplification system, an increase in the amount of nucleic acid chains comprising at least one target sequence takes place. In the second amplification reaction, a portion of this amount may be used as the starting material. This fraction can be added or added to the second reaction mixture, for example. The proportion may for example be relatively low and depends essentially on the requirements of the desired application. Because of the augmenting effect of the PCR, the second amplification may preferably start from more than about 1000 copies of the nucleic acid chains which have been sequence-specifically amplified in the first amplification procedure.

The PCR amplification can thus be started when a sufficient amount of the amplified nucleic acid chains has been synthesized in the first amplification process. In this case, this amount of synthesized nucleic acid chains may include the following ranges: for example, a range of about 10,000 copies to about 100,000,000,000 copies, more preferably from about 10,000 copies to about 1,000,000,000 copies, preferably from about 10,000 to about 100,000,000 copies. In this case, the amount of a target sequence is increased as desired by this first amplification. This increase (based on the starting amount of the target sequence in the batch) can be measured as N times the starting amount and is for example in the following ranges from about 2 times to about 10,000,000,000 times, more preferably from about 10 times to about 1,000,000,000-fold, more preferably from about 10-fold to about 1,00,000,000-fold, preferably from about 10-fold to about 10,000,000-fold, more preferably from about 100-fold to 1,000,000-fold. In this case, based on the volume of the reaction, the following concentration ranges are achieved: from about 10 amol / l to about 10 nmol / l, more preferably from about 1 fmol / l to about 1 nmol / l, even better from about 10 fmol / l to about 0.1 nmol / l, preferably from about 10 fmol / l to about 10 pmol / l.

Since more than 1000 copies of specific nucleic acid chains comprising a target sequence can be produced in the first amplification reaction, a fraction (or aliquot) of this amount can be used for the second amplification reaction.

For example, the PCR may start from an amount including the range of, for example, a range of about 100 copies to about 10,000,000,000 copies, more preferably from about 1,000 copies to about 1,000,000,000 copies, preferably from about 1,000 to about 100,000,000 copies. This amount can be regarded as a subset of the synthesized nucleic acid chain comprising a target sequence increasingly by the first amplification.

Providing amplified synthesis products of the first amplification based on the volume of the reaction, the concentration ranges include: from about 10 amol / l to about 100 nmol / l, more preferably from about 1 fmol / l to about 10 nmol / l, more preferably from about 10 fmol / l to about 1 nmol / l, preferably from about 10 fmol / l to about 10 pmol / l. In this case, a PCR amplification can be started starting from these concentrations of the synthesis products of the first amplification. In the course of the PCR amplification, a further multiplication of products takes place, the final concentration of PCR fragments comprising concentration ranges from 0.01 nmol / l up to 5 μmol / l, better from 1 nmol / l to 1 μmol / l ,

It can be seen that only a fraction of the nucleic acid chains specifically synthesized in the first amplification process can be used in the second amplification process. Thus, in one embodiment, the first amplification provides an amount of copies which represents a relative excess relative to the necessary amount of copies required or desired as starting material for the PCR.

From this first amplification reaction, a portion is transferred to the second amplification reaction, and the synthesized nucleic acid chains contained therein serve as templates for starting the PCR reaction.

A portion of the first reaction mixture can be carried out, for example, by dilution of the first reaction mixture and transfer of a certain volume fraction into the second reaction, this volume including in the first amplification process synthesized nucleic acid chains comprising a target sequence. The proportion of the amount of specifically synthesized nucleic acid chains transferred thereby, which can serve as a template for generating PCR fragments, can comprise the following ranges (calculated on the total amount of synthesized products in the first amplification step): from about 0.0000001% to about 90%, more preferably from about 0.000001% to about 90%, more preferably from about 0.0001% to about 50%, preferably from about 0.001% to about 50%, still more preferably from about 0.01% to about 50%, more preferably 0.1% to about 20%.

This results in not only a dilution of the synthesized nucleic acid chains comprising a target sequence but also the components of the first amplification system, e.g. Primers and controller oligonucleotides. Also possibly formed by-products are diluted. The resulting reduction in the concentration of the components of the first amplification system is accepted and may even have a positive effect on the execution of the PCR. For example, dilution of primers and controller oligonucleotides may result in the oligonucleotide primers or oligonucleotide probes used during the PCR reaction being present in higher concentrations than the transferred oligonucleotide primers of the first amplification system. This may favor the PCR reaction and minimize interference with the amplification process.

Further, dilution of a controller oligonucleotide may also have a positive effect on the PCR reaction by having this controller oligonucleotide initially present during the second amplification reaction, yet its concentration is so reduced during the second amplification that its Binding to the synthesis products or reaction components is slowed down sufficiently or the interaction with complementary sequence segments is reduced or takes place insufficiently and thus its effect on the overall reaction process is reduced by this dilution effect.

Thus, when providing nucleic acid chains for the second amplification, dilution of the reaction components of the first amplification system may also be performed, generally in parallel with dilution of the specific nucleic acid chain provided. The relative amount of components of the first amplification system that are transferred to the second amplification reaction can be detected as a proportion. This proportion refers to the amount of the components used in the first amplification reaction (100%). The transferred proportion of the components of the first amplification system in the second amplification reaction comprises, for example, the following ranges: from about 0.0000001% to about 50%, more preferably from about 0.00000 1% to about 30%, even better from about 0.0001% to about 20%, preferably from about 0.001% to about 10%, more preferably from about 0.1% to about 10%. Thus, the concentrations of the components of the first amplification system are partially significantly reduced, so that their effect in the second amplification step is reduced or seems negligible.

The transferred portion of the first reaction mixture may, for example, relate to the concentrations used or to the volume fractions. For example, the transfer of a microliter (1 μl) of the batch comprising the first amplification system (after completion of amplification) to 49 μl of a batch comprising the second amplification system in a 2% (v / v) dilution or dilution of 1:50 result. Higher dilution levels can be prepared accordingly by means of a dilution series.

For example, the concentration of the controller oligonucleotide in the first amplification system can be reduced from about 10 μmol / L at a dilution of 1: 1000 to about 10 nmol / L. The transferred share is thus only 0.01%. At such a dilution, at the same time, the same proportion of the synthesized nucleic acid chain comprising a target sequence can be transferred. For example, in the first amplification reaction, an amount of about 10,000,000 copies was amplified, and then the transferred amount of products in the PCR reaction at a dilution of 1: 1000 would yield a subset of about 10,000 copies. This initial amount of copies is usually sufficient for the PCR to perform a stable and sufficiently specific amplification reaction.

Also advantageous is the dilution of potentially interfering nucleic acid chains, e.g. of wild-type molecules or allelic variants. During the sequence-specific first amplification, the specific amplification of target molecules takes place predominantly. Other nucleic acid chains are preferably not amplified.

Upon dilution, dilution effect also affects these potentially PCR-interfering sequences. For example, this reduces the amount of wild-type sequences from 100,000 to about 100. This can have a positive effect on the specificity of the PCR reaction.

In a further embodiment, preferably both methods are carried out in one batch (as a "homogeneous assay"). For this purpose, the components of both amplification systems must already be present in one batch at the beginning of the first amplification. The required components for both amplification systems are thus supplied to the reaction mixture before the beginning of the first amplification.

In a preferred embodiment, first the first amplification with required components of the first amplification system takes place in the presence of at least one of the essential components of the second amplification system in the same reaction mixture during the first amplification reaction.

The combination of both amplification systems in a reaction mixture may require adaptation of individual components or concentrations and reaction conditions.

The presence of the components of the second amplification system should not prevent the first amplification reaction.

In one embodiment, the length of the 3 'segment of the third primer which is capable of complementary binding to the controller oligonucleotide is chosen so that this segment does not prevent strand displacement by the controller oligonucleotide. This is essentially achieved by the stability of the double-stranded complex consisting of a controller oligonucleotide and a 3 'segment of the third primer being sufficiently low under reaction conditions of strand displacement by the controller oligonucleotide (eg 65 ° C. step during the first amplification) in that the binding of the third primer to the controller oligonucleotide is only temporary and does not prevent strand displacement. This may be affected, for example, by the length of the 3 'segment of the third primer, which would be able to enter such a complex with the controller oligonucleotide. For example, the length of this complex is between 8 and 20 nucleotides. Furthermore, the 3 'segment of the third primer may have one or more mismatches to sequence composition of the controller oligonucleotide in the corresponding segment, resulting in destabilization of that complex. Preferably, one to three mismatches are positioned in position from -10 to -20 with respect to the 3'-terminal nucleotide of the third primer. This does not significantly affect the primer function of the third primer while reducing the stability of the complex with the controller oligonucleotide.

Furthermore, the controller oligonucleotide from both primers and the polymerase of the second amplification system should not be used as a template. This is particularly important for the third oligonucleotide primer, since this comprises a sequence segment which is complementary to the controller Can bind oligonucleotide. The structure of the controller oligonucleotides in the segment of its possible complementary binding to the third oligonucleotide primer can therefore be designed to be comparable, as in the first primer of the first set. In one embodiment, primer extension of the third oligonucleotide primer is prevented by the controller oligonucleotide, by using nucleotide modifications, substantially preventing the polymerase from extending a third primer bound to the controller oligonucleotide. For example, several 2'-O-alkyl modifications can mediate such blocking activity.

In order to avoid unwanted interference between the two amplification systems, it may furthermore be advantageous to completely or partially convert the components of the second amplification system into an "inert" or "non-active" or "reversibly inactivated" or "non-reactive" state during the first partial amplification vorzuhalten. Only for carrying out the second amplification should such components be converted into an active, i. functional state are transferred.

In one embodiment, at least one of these PCR components is present during the first amplification in a reversibly inactivated form, so that this component does not participate in the first amplification reaction or participation is insignificant. After completion of the first amplification, an activation of these reversibly inactivated components is then carried out, so that the inactivation is canceled and the components are now in active form in the second amplification reaction and can thus perform their role in the amplification.

For example, reversibly inactivated PCR primers or a reversibly inactivated thermostable polymerase (so-called hot-start polymerase) are used. For example, in the conversion step from the first amplification to the second reaction conditions are used which enable activation of this inactive form of the component. In other embodiments, the activation of components occurs continuously under PCR conditions.

Some examples of reversibly inactivated primers are known to one skilled in the art (e.g., termolabile primers called CleanAmp Primers Trilink Technologies). The primers comprise a protected 3'-OH group which can be activated by heating so that the polymerases can start synthesis from this primer. Other examples of reversibly inactivated primers include primers having a 3'-terminal nucleotide comprising a modified sugar residue, e.g. a blocked 3'-OH group (e.g., by a C3 linker at the 3 'position or primers with terminal dideoxy nucleotides, Sambrook et al NAR 1998 p.3073-). When using such primers, it is preferable to use a polymerase for the first amplification which has no 3'-5 'exonuclease activity (e.g., Bst polymerase or its modifications). As a result, such primers remain in the non-activated state during the first phase. When using such primers in the PCR phase, it is thus necessary to activate them. The activation is usually carried out by enzymatic cleavage of the 3'-terminal nucleotide together with the modified sugar residue. For this reason, it is advantageous to use such primers in combination with a thermostable polymerase which comprises a 3'-5 'exonuclease activity (so-called proofreading polymerases). Upon initiation of PCR amplification, such "inactive" primers bind to their complementary positions on templates. After binding to the template, terminal nucleotides, including a blocking group, can be removed by exonuclease activity (which is associated with polymerase). This results in a complementary to the template primer oligonucleotide bound and free 3'-OH groups, which can be extended by polymerase. Such "activated" primers can now be extended by the polymerase so that complementary strands are synthesized to the respective matrices. Polymerases which are capable of being a template-specific activation of such primers under PCR conditions, for example Vent Polymerase, Deep Vent Polymerase, Pfu Polymerase or Phusion Polymerase. By designing PCR primers with a 3'-terminal mismatch to the respective template (or also, for example, in position -1 or -2 relative to the 3'-terminal nucleotide), the activation of such primers can usually be accelerated (eg for Pfu polymerase or Phusion polymerase). Other thermostable polymerases may also cleave modified 3'-OH blocked terminal nucleotides even if they are complementary to the template (e.g., Vent or Deep Vent polymerases). To limit the degradative effect of a polymerase on the PCR primers, such primers may include a 3'-5'-nuclease non-cleavable linkage, e.g. one or more phosphorothioate modifications (PTO modifications) e.g. in positions "minus 2" or "minus 3" or from "minus 2" to "minus 7". This prevents excessive degradation of primers.

The reversibly activated polymerases include, for example, those which are reversibly inactivated by means of an antibody or which are reversibly inactivated by means of a chemical modification (AmpliTaq polymerase). Several commercial suppliers have developed such "hot-start" polymerases for PCR (Qiagen, Thermofisher, Roche). In particular, the Taq polymerase and its modifications in various reversible inactivated states were offered as so-called hot-start Taq polymerase. Particularly preferred are hot-start polymerases, which are converted into an active form, for example by initial heating of the batch to 95 ° C for 1 min to 10 min. Since antibodies can shield different epitopes in polymerases, different activities of polymerases can also be reversibly inactivated in different ways (US Pat. Scalice et al J. Immunol. Methods, 1994, p. 147, Lyamichev et al PNAS, 1999, p. 6143 -)

In one embodiment of the amplification in homogeneous format, the polymerase which has carried out the exponential amplification of the nucleic acid chain to be amplified in the first amplification reaction is inactivated by heating prior to the start of the second amplification. This inactivation can be achieved, for example, by heating the batch to above 80 ° C., better to above 90 ° C., preferably to 95 ° C., for 1 to 10 min. Thus, for example, mesophilic polymerases (such as Bst polymerase or Bsm polymerase, or Gst polymerase) can be inactivated. Such inactivation favors conversion of the process from the first amplification reaction to the second amplification reaction. Thus, at such a temperature step, an inactivation of the polymerase of the first amplification reaction can take place simultaneously and an activation of the polymerase for the second amplification reaction.

Due to such thermal inactivation of the first polymerase, it can no longer bind to the primer or template to the same extent. During the second amplification reaction, therefore, the primer-template complexes are preferably used by the polymerase, which is to carry out the synthesis in the second amplification step.

By such conversion from one polymerase to the other, the other polymerase-associated activities, e.g. Allow strand displacement by Bst polymerase, "turn off", or "turn on" 5'-3 'exonuclease activity of the Taq polymerase.

In a further embodiment, during the second amplification reaction, the 5 'segment of the controller oligonucleotide hybridized to the first primer extension product can be cleaved by the 5'-3 nuclease of the polymerase (eg 5'-3' nuclease of a Taq polymerase). This process is well known and is often used for probe degradation and real-time monitoring of a PCR reaction ( US Pat 5,210,015 ). In this case, the polymerase can complementarily extend the fourth PCR primer using the third primer extension product under suitable reaction conditions by incorporation of nucleotides and at the same time enzymatically cleaving the controller oligonucleotide in its 5 'segment of the third region hybridized to the third primer extension product. The polymerase-extended strand preferably forms an overlap with the controller oligonucleotide around at least one 3'-terminal nucleotide during synthesis, thereby forming a structure recognizable for 5'-3'-nuclease activity of the Taq polymerase under used reaction conditions, and this structure can be split. By 5'-3 'exonuclease activity cleavage of the phosphodiester bonds occurs between nucleotides of the third region of the controller oligonucleotide. The 5'-3'-exonuclease-related cleavage occurs preferentially on the DNA strand, with some sugar-phosphate backbone modifications, such as 2'-OMe ribonucleotides or PTO modifications or PNA, slowing down or even preventing this cleavage. For this reason, a combination of a polymerase comprising a 5'-3 'exonuclease activity (eg tag polymerase) and a controller oligonucleotide is preferably used in this embodiment, wherein the controller oligonucleotide in its 5' segment of the third Area, predominantly by nuclease cleavable nucleotides or their analogs includes (eg DNA). The length of this 5 'segment of the controller oligonucleotides comprises, for example, 5 to 50, preferably 10 to 30 nucleotides.

By degrading the controller oligonucleotide bound to the first primer extension product, a polymerase, even without strand displacement properties, can extend the second primer and thus synthesize the second primer extension product.

In one embodiment, the presence of components of the second amplification system during the first amplification is considered as follows:

On the one hand, the effect of the components of the second amplification system can be reduced by their reversible inactivation prior to the first amplification. On the other hand, individual sequence elements should not be considered nonspecific templates for primer extension during the first amplification occur. For this reason, primers of the first and second amplification systems should be checked for the existence of self-complementary structures in order to avoid primer-dimer formation.

The third PCR primer typically comprises a sequence segment in its 3 'segment which comprises regions complementary to the controller oligonucleotide. This allows the third PCR primer to bind to the controller oligonucleotide complementary. To perform the first amplification, the third PCR primer must not prevent strand displacement by the controller oligonucleotide during the first amplification. For this purpose, the sequence length or sequence composition of the 3 'segment of the third PCR primer is adjusted so that it does not substantially bind to the controller oligonucleotide under reaction conditions of the strand displacement step (e.g., 65 ° C). This can be achieved, for example, by the melting temperature of the complex of a controller oligonucleotide and the 3 'segment of the third primer being substantially below the reaction temperature of the strand displacement step, e.g. between 45 ° and 60 ° C. Such a third PCR primer may further bind with its 3 'segment to its template and initiate a primer extension reaction. This may be achieved, for example, by the 3 'segment of the third PCR primer, which may be complementary to the controller oligonucleotide, comprising in its length regions of between 9 and 30 nucleotides, more preferably between 12 and 20 nucleotides , preferably between 12 and 16 nucleotides. This 3 'segment of the third PCR primer may comprise one or more mismatches to corresponding positions of the controller oligonucleotide so that the binding strength continues to decrease.

The sequence segment of the controller oligonucleotide which can substantially complementarily bind the third PCR primer is referred to as the fourth region of the controller oligonucleotide. This fourth region of the controller oligonucleotide may include sequence segments of the second region and / or the third region.

The fourth region of the controller oligonucleotide comprises in its length regions which are between 9 and 30 nucleotides, more preferably between 12 and 20 nucleotides, preferably between 12 and 16 nucleotides. The fourth region of the controller oligonucleotide may comprise one or more mismatches to the 3 'segment of the third PCR primer. Preferably, this fourth region comprises nucleotide modifications that prevent the third PCR primer from being extended by a polymerase of the first and / or second amplification system using the controller oligonucleotide as a template. Such modifications have already been described for the combination of the first primer and the controller oligonucleotide. They include, for example, sequence segments with 2'-O-alkyl modifications, wherein the length of this segment may be between 6 and 30 modifications and the complementary nucleotide of the controller oligonucleotide to the 3'-terminal nucleotide of the third PCR primer preferably itself such a modification and further flanked on both sides by at least three nucleotides with such modifications. This ensures that the controller oligonucleotide from the third and fourth primers and the polymerase of the second amplification system is not used as a template.

The creation of the first and second primer extension product of a defined length plays a role in the separation of these two products by means of a controller oligonucleotide. For this reason, it is advantageous to prevent any premature extension of 3 'ends (each of the first and second primer extension products) using a third or fourth primer of the second amplification system as a template during the first amplification reaction.

A reduction or prevention of a possibly unwanted excess extension of the 3 'end of the first primer extension product using the fourth PCR primer as a template can be achieved in various ways: In one embodiment, this is achieved by the fourth PCR primer, when attached to the 3 'segment of the first primer extension product, can not undergo perfect match complementary binding with the 3'-terminal nucleotide of this synthesized first primer extension product. This is intended to prevent or at least reduce excess extension of the 3'-terminal nucleotides of the first primer extension product.

In a further embodiment, this is achieved in that the fourth PCR primer, when attached to the 3 'segment of the first primer extension product, is unable to undergo perfect match complementary binding with the at least one of the terminal nucleotides of that synthesized first primer extension product , The resulting at least one mismatch position is preferably from in the position of - 1 to - 5 relative to the terminal nucleotide of the first primer extension product.

A reduction or prevention of a possibly unwanted excess extension of the 3 'end of the second primer extension product using the third PCR primer as a template can be achieved in various ways: In one embodiment, this is achieved by the third PCR primer, when bound to the 3 'segment of the second primer extension product, can not undergo perfect match complementary binding with the 3'-terminal nucleotide of this synthesized first primer extension product. This is intended to prevent or at least reduce excess extension of the 3'-terminal nucleotides of the second primer extension product.

In a further embodiment, this is achieved in that the third PCR primer, when attached to the 3 'segment of the second primer extension product, can not undergo perfect match complementary binding with the at least one of the terminal nucleotides of this synthesized second primer extension product , The resulting at least one mismatch position is preferably in the position of -1 to -5 relative to the terminal nucleotide of the second primer extension product.

The presence of components of the first amplification system during the second amplification is considered as follows:

On the one hand, the effect of the components of the first amplification system can be reduced by their dilution prior to the second amplification. On the other hand, individual elements should not occur as nonspecific templates for primer extension during the second amplification. For this reason, the primers of the first and second amplification systems should be checked for the presence of self-complementary structures in order to avoid primer-dimer formation.

In a preferred embodiment, the second amplification is carried out in the presence of the controller oligonucleotide of the first amplification system. For this reason, the design of the controller oligonucleotide, the primers of the second set and the reaction conditions must be adjusted so that the second amplification reaction is not significantly disturbed by the presence of the controller oligonucleotide.

First, the controller oligonucleotide from both primers and the polymerase from the second amplification system should not be used as a template. This is particularly important for the third oligonucleotide primer, since it comprises a sequence segment which can bind in a complementary manner to the controller oligonucleotide in the fourth region. The structure of the controller oligonucleotides in the fourth region preferably comprises modification which prevents potential primer extension. For example, several 2'-O-alkyl modifications can mediate such a synthetic blocking effect.

The controller oligonucleotide may form on the third primer extension product during the second amplification and thus a complementary strand with the third primer extension product. Such a double-stranded fragment may possibly prevent a polymerase from starting to synthesize the complementary strand to the full extent starting from the fourth PCR primer, up to and including the 5 'segment of the third primer extension product. Therefore, polymerase selection and choice of reaction conditions should be designed so that binding of the controller oligonucleotides does not prevent synthesis of the fourth primer extension product.

The length of the segment of the third primer extension product which is capable of complementary binding with the controller oligonucleotide is co-determined, for example, by the positioning of the third primer. Preferably, the third primer is arranged so that the length of the resulting fully complementary portion of an expected third primer extension product to the controller oligonucleotide is preferably in the range of from about 15 nucleotides to about 60 nucleotides, more preferably from about 20 nucleotides to about 40 nucleotides , more preferably from about 20 nucleotides to about 30 nucleotides.

In one embodiment, a thermostable polymerase is preferably selected in the second amplification reaction comprising strand displacement activity, for example, a thermostable polymerase such as Vent Exo minus polymerase or pyrophage polymerase can be used. This allows the controller oligonucleotide to be separated from the third primer extension product during the second amplification, and the fourth primer extension product can be synthesized in full length.

In a further embodiment, a polymerase is preferably selected in the second amplification reaction which is capable of expressing the controller oligonucleotide by 5'-3 'exonuclease activity of the Polymerase to cleave, with a simultaneous primer extension takes place (see above). In this case, a controller oligonucleotide is used, which can be cleaved by a 5'-3 'exonuclease, at least in its 5' segment. The degradation progressively leads to a shortening of the hybridized to the third primer extension product controller oligonucleotide, which under suitable reaction conditions (eg, temperature of about 65 ° C to 75 ° C) to destabilize this bond and finally to a separation of the bond to the third primer extension product leads. This allows the fourth full-length primer extension product to be synthesized.

In another embodiment, the concentration of the controller oligonucleotide and the concentration of the fourth primer of the second set of primers are chosen such that the primer binding and the primer extension are faster under the chosen reaction conditions than the binding of the controller oligonucleotides the third primer extension product. For example, concentrations of the controller oligonucleotide in the range of 0.01 μmol / L to 0.3 μmol / L are used. Concurrently, concentrations of the fourth primer are used which range from about 0.5 μmol / L to 2 μmol / L. Preferably, polymerases are used in the second amplification reaction which have high processivity (e.g., phage polymerase). The binding of the primers and their extension are usually faster than the binding of the controller oligonucleotides, so that the primer extension reaction is kinetically advantageous.

In a further embodiment, the primer extension reaction is carried out in the second amplification reaction at at least two temperatures. At the first, generally lower temperature, e.g. in the range of 45 ° C to 65 ° C, the complementary binding of the fourth primer to its complementary segment occurs in the 3 'segment of the third primer extension product, as well as an initial extension by the polymerase. Here, a partially extended primer extension product is generated which has sufficient stability with the third primer extension product such that after increasing the temperature of the second region, e.g. to levels of about 70 ° C to about 80 ° C, this partially extended fourth primer extension product can be extended by polymerase. At this temperature, the controller oligonucleotide can spontaneously dissociate from its binding with the third primer extension product so that the polymerase can continue the synthesis of the fourth primer extension product, up to and including the 5 'segment of the third primer extension product copied from the polymerase becomes. Preferably, polymerases are used in the second amplification reaction which have high processivity (e.g., phage polymerase).

Methods with the composition of the reaction mixture in the "homogeneous format" are particularly suitable, for example, if a division of the first reaction mixture is not useful or technically very complicated, for example, in a "micro- or nanotube-reaction" (as well as digital PCR called).

Likewise, further elements can be supplied to an amplification, for example a solid phase comprising oligonucleotides which are suitable for a specific binding of products formed and / or also occur as immobilized primers, so that a solid phase PCR can be carried out, the primers Extension products are immobilized on the solid phase. The establishment of contact with a solid phase comprising specific oligonucleotides may occur before, during, or only after the first amplification.

The reaction mixture can be divided into a plurality of reaction volumes (partitioning) before the start of the first amplification reaction, so that an amplification of a reaction mixture is simultaneously divided into about 100 or 1000 or more equal volume fractions. This division can take place in the form of droplets (for example as an emulsion). The execution of amplification can be performed in parallel on this plurality of droplets (e.g., as Digital PCR).

The sequence of the amplification reactions (first a sequence-specific amplification and only then a PCR) plays an essential role: the sequence-specific amplification (first amplification) using a controller oligonucleotides is preferably carried out before a PCR amplification. In order to avoid PCR-based distortions in the target sequences and side reactions before a first amplification. Therefore, the PCR-based amplification takes place only as a second step.

In a preferred embodiment, therefore, for the first amplification reaction no DNA fragments generated by a PCR or another amplification method are used as starting nucleic acid chains.

The first amplification method (first partial amplification) and the required components of the first amplification system will first be described schematically, then the second amplification method (second partial amplification) and the components of the second amplification system will be described. Subsequently, combination of the first and second amplification methods, as well as advantageous embodiments will be presented. The second amplification method (also referred to below as PCR) takes place after the first amplification.

Preferably, the first amplification occurs as an exponential amplification in which newly synthesized products of both primers (primer extension products) occur as templates for further synthetic steps. In this case, the primer sequences are at least partially copied, so that complementary primer binding sites arise, which are present immediately after their synthesis as sequence segments of a double strand. In the first amplification process, synthesis steps of both strands and double-strand opening steps of the newly synthesized sequence segments take place in mutual alternation. Sufficient double-strand separation after synthesis is an important prerequisite for further synthesis. Overall, such a change from synthesis and double-strand separation steps can lead to exponential amplification.

In the first amplification process according to the invention, the double-stranded opening of the main products of the amplification (amplification of nucleic acid chains comprising target sequence) takes place inter alia by means of an oligonucleotide, termed controller oligonucleotide. The controller oligonucleotide preferably comprises sequence segments corresponding to the target sequence.

In detail, the strand separation is achieved according to the invention by using controller oligonucleotides with predefined sequences, which preferably separate a newly synthesized double strand consisting of two specific primer extension products by means of a sequence-dependent nucleic acid-mediated strand displacement. The resulting single-stranded segments of the primer extension products comprise the target sequence as well as corresponding primer binding sites, which can serve as binding sites for further primer oligonucleotides, so that an exponential amplification of nucleic acid chains to be amplified is achieved. The primer extension reactions and strand displacement reactions preferably take place simultaneously in the batch. The amplification is preferably carried out under reaction conditions which do not permit spontaneous separation of both specific synthesized primer extension products.

A specific exponential first amplification of a nucleic acid sequence comprising a target sequence comprises a repetition of synthesis steps and double-strand opening steps (activation steps for primer binding sites) as a mandatory prerequisite for the amplification of the nucleic acid chain.

The opening of synthesized duplexes is implemented as a reaction step which is to be sequence-specifically influenced by the controller oligonucleotide. This opening can be complete, even to the dissociation of both complementary primer extension products, or even partial.

According to the invention, the controller oligonucleotide comprises sequence segments which can interact with the target sequence and further sequence segments which bring about this interaction or facilitate or favor it. In the context of the interaction with the controller oligonucleotide, double-stranded portions of the synthesized primer extension products are converted into a single-stranded form via sequence-specific strand displacement. This process is sequence-dependent: only when the sequence of the synthesized duplex has some degree of complementarity with the corresponding sequence of the controller oligonucleotide will there be sufficient double-stranded opening such that the sequence segments essential to the continuation of the synthesis, e.g. Primer binding sites are converted into single-stranded form, which corresponds to an "active state". The controller oligonucleotide thus "specifically" activates the newly synthesized primer extension products comprising the target sequence for further synthetic steps.

In contrast, sequence segments which do not comprise a target sequence are not converted into a single-stranded state and remain as a double strand, which corresponds to an "inactive" state. The potential primer binding sites in such a duplex are less favored or completely hindered from interacting with new primers, so that further synthetic steps on such "non-activated" strands generally do not occur. This missing or diminished activation (ie transfer into one single-stranded state) of synthesized nucleic acid strands after a synthesis step results in that in the subsequent synthesis step only a reduced amount of primers can successfully participate in a primer extension reaction.

Due to a desirable exponential first amplification of major products (a nucleic acid sequence to be amplified comprising target sequences) several synthesis steps and activation steps (double-stranded opening steps) are combined to an amplification process and carried out as many times or repeated until the desired Amount of the specific nucleic acid chain is provided.

The reaction conditions (e.g., temperature) are designed in the course of the first amplification process so that spontaneous separation of complementary primer extension products in the absence of a controller oligonucleotide is unlikely or significantly slowed.

The desired enhancement of specificity of amplification thus results from the sequence dependence of the separation of complementary primer extension products comprising a target sequence: the controller oligonucleotide facilitates this double-stranded separation as a result of matching its sequence segments to given sequence segments of the primer extension products , This match is checked after each synthesis cycle by the controller oligonucleotide. The exponential amplification results from successful repeats of syntheses and sequence-specific strand displacements caused by controller oligonucleotide, i. "Activations" (double-stranded openings / double-stranded separations / strand displacement processes resulting in single-stranded form of corresponding primer binding sites) of newly synthesized primer extension products.

The use of a pre-defined controller oligonucleotide thus allows a sequence-dependent review of the contents of the primer extension products between the individual synthetic steps during the exponential amplification and inducing a selection of sequences for subsequent synthetic steps. Here, "active", single-stranded states of newly synthesized specific primer extension products may result as a result of successful interaction with a controller oligonucleotide, and "inactive" double-stranded states of newly synthesized non-specific primer extension products as a result of deficient, insufficient, decreased and / or slowed down Interaction with a controller oligonucleotide can be distinguished.

For exponential amplification the following effects result:

Under non-denaturing conditions, the separation of specifically synthesized strands takes place with the aid of controller oligonucleotide.

The exponential amplification of nucleic acid chains comprising a target sequence is carried out in a sequence-controlled manner (main reaction). This sequence control occurs after each step in the synthesis and includes sequence segments which are between the primers and comprise a target sequence. The successful verification of the result of the synthesis after each synthesis step results in separation of the two specific primer extension products, which is the prerequisite for further specific synthesis steps.

During the first amplification, an initial generation or generation of unspecific primer extension products can not be ruled out in principle (by-products). Due to a template dependency, such non-specific primer extension products are usually present in double-stranded form immediately after their synthesis. However, the interaction with the controller oligonucleotide either remains completely or is restricted so that strand separation does not occur or strand separation is slower than the main reaction. The transmission of erroneous sequence information from one synthesis cycle to the next does not take place.

By choosing the reaction conditions and the design of the controller oligonucleotide, it is thus possible to exert a targeted influence on the efficiency of the regeneration of correct nucleic acid chain templates between individual synthesis steps in an amplification process. In general, the higher the degree of correspondence of the synthesized sequence to the given sequence of the controller oligonucleotide, the more successful the separation of the synthesized products, the more successful the regeneration of correct templates from one synthesis step to the next. Conversely, sequence variation in by-products results in insufficient regeneration of template strands and thus slowing of the synthesis initiation or reduction of yield in each subsequent cycle. All exponential amplification of by-products either proceeds more slowly or does not occur at all and / or remains at an undetectable level.

The method thus allows a review of the synthesized sequences in real time, i. without reaction disruption and thus represents a potential for the development of homogeneous assays, in which all components of the assay already exist at the beginning of a reaction in the reaction mixture.

As a result of the first partial amplification, a sufficient amount of highly specific amplification fragments is provided, which can serve as template in the second partial amplification (PCR).

After a sufficient reaction time or cycle number, the first amplification process is then terminated or the reaction conditions are changed so that the second amplification process (PCR) can be started. In the second amplification method, the amplification of the target sequences or their portion is carried out using nucleic acid chains prepared in the first amplification method and at least one other, PCR-specific components (e.g., an oligonucleotide primer or a PCR polymerase). Since the number of specific nucleic acid chains is sufficiently high already at the beginning of the second amplification, the PCR process requires fewer cycles until the desired total amount of products is reached. This PCR products are generated, which have fewer errors overall.

The sequential switching of a highly specific first amplification method at the beginning of the amplification and the PCR only after this, thus the overall specificity of the generated PCR products can be improved, in comparison with conventional PCR methods which operate from the beginning under PCR conditions.

Preferably, components for both amplification methods are already present at the beginning of the amplification.

In one embodiment, the components of the first amplification system differ from components of the second amplification system (PCR).

In one embodiment, the components of the first amplification system are different from components of the second amplification system and the PCR components are under reaction conditions of the first amplification process at least partially in non-active form (e.g., as hot-start polymerases). Switching from the first amplification to the second amplification thus requires an additional activation step of the PCR components (e.g., polymerase).

In a further embodiment, the polymerase of the first amplification system is inactivated after completion of the first amplification, so that a different polymerase is used for the second amplification (PCR).

In a further embodiment, the components of the first amplification system are also partially used by the second amplification system, wherein at least one further PCR-specific component (eg a further primer oligonucleotide or a polymerase) is used, which are not part of the first amplification system ,

The present invention describes some embodiments of both methods, arrays of oligonucleotide primers, which are advantageous for performing both amplification methods. By suitable arrangement of individual elements of the first and second amplification systems on a nucleic acid chain comprising a target sequence, homogeneous assays with higher total specificity can be assembled.

terms and definitions

In the context of the present invention, the terms used have the following meanings:

The term "oligonucleotide" as used herein with reference to primer, controller oligonucleotide, probes, nucleic acid chain to be amplified is defined as a molecule having two or more, preferably comprises more than three deoxyribonucleotides and / or ribonucleotides and / or nucleotide modifications and / or non-nucleotide modifications. Its length includes, for example, ranges between 3 to 300 nucleotide units or its analogs, preferably between 5 to 200 nucleotide units or its analogs. Its exact size depends on many factors, which in turn depend on the ultimate function or use of the oligonucleotides.

As used herein, the term "primer" refers to an oligonucleotide, whether naturally occurring e.g. B. occurs in a purified restriction cleavage or was produced synthetically. A primer is capable of acting as the initiation point of the synthesis when used under conditions which induce the synthesis of a primer extension product complementary to a nucleic acid strand, i. H. in the presence of nucleotides and an inducing agent, e.g. DNA polymerase at a suitable temperature and pH. The primer is preferably single-stranded for maximum efficiency in amplification. The primer must be long enough to initiate synthesis of the extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including reaction temperature and primer source and application of the method. For example, the length of the oligonucleotide primer in diagnostic applications, depending on the complexity of the target sequence between 5 to 100 nucleotides, preferably 6 to 40 and particularly preferably 7 to 30 nucleotides. Short primer molecules generally require lower reaction temperatures to perform their primer function to form sufficiently stable complexes with the template or higher concentrations of other reaction components, such as DNA polymerases, such that sufficient elongation of formed primers is achieved. Template complexes can be done.

The primers used herein are selected to be "substantially" complementary to the different strands of each specific sequence to be amplified. That is, the primers must be sufficiently complementary to hybridize with their respective strands and initiate a primer extension reaction. Thus, for example, the primer sequence does not need to reflect the exact sequence of the target sequence. For example, a non-complementary nucleotide fragment may be attached to the 5 'end of the primer, with the remainder of the primer sequence being complementary to the strand. In another embodiment, single non-complementary bases or longer non-complementary sequences may be inserted in a primer, provided that the primer sequence has a sufficiently large complementarity with the strand sequence to be amplified to hybridize therewith and thus a primer template -Complex able to produce the synthesis of the extension product.

As part of the enzymatic synthesis of a template-complementary strand, a primer extension product is generated which is completely complementary to the template strand.

Tm melting temperature

The melting temperature of a complementary or partially complementary double strand is generally understood to mean a measured value of a reaction temperature at which approximately half of the strands are in the form of a double strand and the other half is in the form of a single strand. The system (association and dissociation of strands) is in equilibrium.

Due to a large number of factors which can influence the Tm of a double strand (eg sequence length, CG content of the sequence, buffer conditions, concentration of divalent metal cations etc.), the determination of the Tm of a nucleic acid to be amplified should take place under the same conditions, like the intended amplification reaction.

Because of the dependence of the measurable melting temperature on multiple reaction parameters, e.g. of respective buffer conditions and respective concentrations of the reactants, melting temperature is understood to mean a value which was measured in the same reaction buffer as the exponential amplification, at concentrations of both complementary components of a double strand of about 0.1 μmol / l to about 10 μmol / 1, preferably in concentration from about 0.3 μmol / to about 3 μmol / l, preferably at about 1 μmol / l. The respective value of the melting temperature is a guideline, which correlates with the stability of a respective double strand.

The deoxyribonucleoside triphosphates (dNTPs) dATP, dCTP, dGTP and TTP (or dUTP, or dUTP / TTP mixture) are added in adequate amounts to the synthesis mixture. In one embodiment, in addition to dNTPs, at least one other type of dNTP analogs can be added to the synthesis mixture. These dNTP analogs in one embodiment include, for example, a characteristic Labeling (eg, biotin or fluorescent dye), so that when incorporated into nucleic acid strand, this label is integrated into the nucleic acid strand. In another embodiment, these dNTP analogs comprise at least one modification of the sugar-phosphate portion of the nucleotide, eg, alpha-phosphorothioate-2'-deoxyribonucleoside triphosphates (or other modifications which confer nuclease resistance to a nucleic acid strand), 2 ', 3'-Dideoxy ribonucleoside triphosphates, acyclo nucleoside triphosphates (or other modifications leading to termination of synthesis). In a further embodiment, these dNTP analogs comprise at least one modification of nucleobase, eg iso-cytosines, iso-guanosines (or other modifications of the nucleobases of the extended genetic alphabet), 2-amino-adenosines, 2-thiouridines, inosines, 7 deazy-adenosines, 7-deaza-guanosines, 5-Me-cytosines, 5-propyl-uridines, 5-propyl-cytosines (or other modifications of nucleobases that can be incorporated into natural nucleobases by a polymerase and alter the strand Lead stability). In another embodiment, a dNTP analog comprises both a modification of the nucleobase and a modification of the sugar-phosphate moiety. In a further embodiment, at least one further type of dNTP analogue is added to the synthesis mixture instead of at least one natural dNTP substrate.

The nucleic acid synthesis inducing agent may be an enzyme entrapping compound or a system that functions to effect the synthesis of the primer extension products.

Suitable enzymes for the first amplification for this purpose include e.g. DNA polymerases such as Bst polymerase and its modifications, Vent polymerase and others - preferably heat stable DNA polymerases which allow the incorporation of the nucleotides in the correct manner, thereby forming the primer extension products that are complementary to any nucleic acid strand synthesized. Generally, the synthesis is initiated at the 3 'end of each primer and then proceeds in the 5' direction along the template strand until the synthesis is complete or interrupted.

Preferably, template-dependent DNA polymerases are used in the first amplification which are capable of strand displacement. These include, for example, a large fragment of Bst polymerase or its modifications (e.g., Bst 2.0 DNA polymerase), Klenow fragment, Vent exo minus polymerase, Deepvent exo minus DNA polymerase, large fragment of Bsu DNA polymerase, large fragment of Bsm DNA polymerase.

In one embodiment, preference is given to using polymerases which have no 5'-3'-exonuclease activity or have no 5'-3'-FEN activity.

For the second amplification thermostable matrix-dependent DNA polymerases are preferably used. For example, Taq polymerase or its modifications (e.g., Ampli-Taq), or proofreading polymerases: Pfu and its modifications or Vent Polymerase and its modifications. Polymerases may be used which have been fused with another protein to increase their processivity, e.g. Phusion polymerase. A variety of polymerases are commercially available.

Combinations of polymerases may also be used, e.g. OneTaq polymerase (NEB) is a combination of a Taq polymerase and Vent polymerase. Such combinations of polymerases can contribute to higher synthesis accuracy in the second amplification.

In one embodiment, at least two different polymerases are used, for example polymerases which are capable of strand displacement and those which have a 3'-5 'proofreading activity.

In preferred embodiments, polymerases are used with a hot-start function, which can only perform their function when a certain temperature is reached.

First amplification system includes components necessary to perform a specific first amplification. These include, for example, the first primer oligonucleotide, the second primer oligonucleotide, the controller oligonucleotide and the first polymerase.

In addition, nucleotide mixtures and buffer systems may also be included to the extent necessary to perform a specific first amplification (e.g., specific buffer systems for polymerase).

The first amplification system supports synthesis of the first amplification fragment 1.1 (or the first amplification product 1.1 also referred to as the first amplification nucleic acid chain to be amplified) during the first amplification (also called first partial amplification).

Second amplification system includes components necessary to perform a specific second amplification. These include, for example, the third primer oligonucleotide, the fourth primer oligonucleotide and the second polymerase.

In addition, nucleotide mixtures and buffer systems may be included to the extent necessary to perform a specific second amplification (e.g., specific buffer systems for polymerase).

The second amplification system supports synthesis of the second amplification fragment 2.1 (or the second amplification product 2.1 ) during the second amplification (also called second partial amplification).

First primer oligonucleotide:

(Component of the first amplification system)

The first primer oligonucleotide ( 12 to 16 ) includes a first primer region and a second region. The first primer region is capable of binding to a substantially complementary sequence within the nucleic acid or its equivalents to be amplified and initiating a primer extension reaction. The second region comprises a polynucleotide tail which is capable of binding a controller oligonucleotide and thereby effecting spatial proximity between the controller oligonucleotide and the other portions of the first primer extension product sufficient to induce strand displacement through the controller oligonucleotide. The second region of the first primer oligonucleotide further comprises at least one modification (a nucleotide modification or a non-nucleotide modification) which prevents the polymerase from copying the polynucleotide tail by inhibiting the continuation of polymerase dependent synthesis. This modification is located, for example, at the junction between the first and second regions of the first primer oligonucleotide. The first primer region of the first primer oligonucleotide is thus replicable by a polymerase such that a complementary sequence to this region can be generated during the synthesis of the second primer extension product from the polymerase. The polynucleotide tail of the second region of the first primer oligonucleotide is preferably not copied by the polymerase. In one embodiment, this is achieved by the modification in the second region, which stops the polymerase from the polynucleotide tail. In another embodiment, this is accomplished by nucleotide modifications in the second region, where the entire polynucleotide tail consists essentially of such nucleotide modifications and thus is non-cleavable for polymerase.

In one embodiment, each first primer oligonucleotide is specific for each nucleic acid to be amplified.

In one embodiment, each first primer oligonucleotide is specific for at least two of the nucleic acids to be amplified, each comprising substantially different sequences.

In one embodiment, the first primer oligonucleotide is labeled with a characteristic marker, e.g. a fluorescent dye (e.g., TAMRA, fluorescein, Cy3, Cy5) or an affinity tag (e.g., biotin, digoxigenin) or an additional sequence fragment, e.g. for binding a specific oligonucleotide probe for detection or immobilization or barcode labeling.

A first primer oligonucleotide is preferably used as a primer in the first amplification.

In another embodiment, it may be used as a primer in both the first and second amplifications.

Second primer oligonucleotide:

(Component of the first amplification system)

An oligonucleotide capable of binding with its 3 'segment to a substantially complementary sequence within the nucleic acid or its equivalents to be amplified and initiating a specific second primer extension reaction. This second primer oligonucleotide is thus capable of binding to the 3 'segment of a first specific primer extension product of the first primer oligonucleotide and initiating polymerase-dependent synthesis of a second primer extension product.

The length of the second primer oligonucleotide may be between 15 and 100 nucleotides, preferably between 20 and 60 nucleotides, more preferably between 30 and 50 nucleotides.

In one embodiment, every second primer oligonucleotide is specific for each nucleic acid to be amplified.

In one embodiment, every second primer oligonucleotide is specific for at least two of the nucleic acids to be amplified, each comprising substantially different sequences.

In one embodiment, the second primer oligonucleotide is labeled with a characteristic marker, e.g. a fluorescent dye (e.g., TAMRA, fluorescein, Cy3, Cy5) or an affinity tag (e.g., biotin, digoxigenin) or an additional sequence fragment, e.g. for binding a specific oligonucleotide probe for detection or immobilization or barcode labeling.

A second primer oligonucleotide is preferably used as a primer in the first amplification. In another embodiment, it may be used as a primer in both the first and second amplifications.

Primer extension product:

A primer extension product (also called a primer extension product) results from enzymatic (polymerase-dependent) extension of a primer oligonucleotide as a result of template-dependent synthesis catalyzed by a polymerase.

A primer extension product comprises the sequence of the primer oligonucleotide in its 5 'segment and the sequence of the extension product (also called extension product) which has been synthesized by a polymerase in a template-dependent form. The extension product synthesized by the polymerase is complementary to the template strand on which it was synthesized.

A specific primer extension product (eg P1.1 -Ext and P2.1 -Ext) of the first amplification (main product) comprises sequences of the nucleic acid chain to be amplified. It is the result of a specific synthesis or a correct execution of an intended primer extension reaction in which the nucleic acid chain to be specifically amplified serves as a template. In a preferred embodiment, the sequence of the synthesized primer extension products coincides completely with the expected sequence of a nucleic acid to be amplified. In another embodiment, deviations in the sequence obtained may be tolerated by the theoretically expected sequence. In one embodiment, the extent of correspondence of the sequence obtained due to amplification with the sequence of the theoretically expected nucleic acid to be amplified is for example between 90% and 100%, preferably the agreement is greater than 95%, ideally the agreement is greater than 98% (measured at the proportion of synthesized bases).

The length of the extension product of a specific primer extension product may be between 10 and 300 nucleotides, more preferably between 10 and 180 nucleotides, preferably between 20 and 120 nucleotides, more preferably between 30 and 80 nucleotides.

For example, in the second amplification, products of a specific template-dependent primer extension reaction of the third and fourth primers represent the major products or intermediates: the partial third primer extension product ( P3.1 -ext part 1 . 21 ), or the complete primer extension product ( P3 .1-Ext, 21 ), the partial fourth primer extension product ( P4.1 -Ext part 1 . 21 ), or the complete primer extension product ( P4.1 -ext, 21 ). These products in one embodiment comprise a target sequence or its equivalents. In another embodiment, the second primer extension product comprises a target sequence and the first primer extension product comprises equivalents of the target sequence, namely the complementary strand. In a further embodiment, the fourth primer extension product comprises a target sequence and the third primer extension product comprises equivalents of the target sequence, namely the complementary strand. In another embodiment, the fourth primer extension product comprises portions of a target sequence and the third primer extension product comprises equivalents of these portions of the target sequence, namely the complementary strand.

The first primer extension product ( P1.1 -Ext) and the second primer extension product ( P2.1 -Ext) together make up the first amplification fragment 1.1 (also as amplification product 1.1 ) of the first amplification ( 1 ).

The third primer extension product ( P3.1 -Ext) and the fourth primer extension product ( P4.1 -Ext) together make up the second amplification fragment 2.1 (Also as the second amplification product 2.1 ) of the second amplification ( 1 ).

The third primer extension product ( P3.1 -Ext) may also be referred to as a complete third primer extension product. This product is formed using the fourth Primr extension product ( P4.1 -Ext) as a template. The fourth primer extension product ( P4.1 Text) may also be referred to as a complete fourth primer extension product. This product is made using the third Primr extension product ( P3.1 -Ext) as a template ( 21 ).

In contrast, a partial third primer extension product ( P3.1 Extract Part 1) using the second Primr extension product ( P2.1 -Ext) is generated as a template as an intermediate. The partial fourth primer extension product ( P4.1 Extract Part 1) using the first primer extension product ( P1.1 -Ext) generated as a template as an intermediate ( 21 )

For example, a non-specific primer extension product (by-product) includes sequences that have resulted as a result of a nonspecific or improper primer extension reaction. These include, for example, primer extension products that have arisen as a result of a false initiation event or as a result of other side reactions, including polymerase-dependent sequence changes such as base substitution, deletion, etc. The level of sequence aberrations of nonspecific primer extension products generally exceeds the ability of controller oligonucleotides to successfully displace such double-stranded by-products from their templates so that amplification of such by-products is slower or completely absent. The extent of acceptance or the tolerance limit for deviations depend, for example, on the reaction temperature and the manner of sequencing deviation. Examples of nonspecific primer extension products are primer dimers or sequence variants which do not correspond to the nucleic acid to be amplified, e.g. Sequences which do not comprise a target sequence.

The assessment of a sufficient specificity of the amplification is often related to the task. In many amplification methods, some degree of unspecificity of the amplification reaction can be tolerated as long as the desired result can be achieved. In a preferred embodiment, the proportion of nucleic acid chains to be amplified in the overall result of the reaction is more than 1%, more preferably more than 10%, even more preferably more than 30%, based on the total amount of newly synthesized strands.

The nucleic acid to be amplified

(expected result of the first amplification)

The nucleic acid to be amplified represents a nucleic acid chain which is to be amplified sequence-specifically or at least predominantly sequence-specifically by means of the first amplification using primers and controller oligonucleotide by the polymerase. The nucleic acid to be amplified comprises both strands of the first amplification fragments 1.1 (also as the first amplification product 1.1 indicated) ( 1 ). It can be used as a start-up nucleic acid chain 2.1 can be used, wherein at least one of their strands can be used to initiate the second amplification.

The length of the nucleic acid to be amplified may be between 20 and 300 nucleotides, more preferably between 30 and 200 nucleotides, preferably between 40 and 150 nucleotides, particularly preferably between 50 and 100 nucleotides.

The nucleic acid sequence to be amplified may comprise one or more target sequences or their equivalents. Furthermore, a nucleic acid to be amplified may comprise sequences substantially complementary to a target sequence which are propagated with similar efficiency as a target sequence in an amplification reaction and comprise target sequence or its segments. In addition to a target sequence, the nucleic acid to be amplified may include additional sequence segments, for example, primer sequences, sequences with primer binding sites and / or sequence segments for binding of detection probes, and / or sequence segments for sequence encoding of strands by barcode sequences and / or sequence segments for binding to a solid phase. The primer sequences or their sequence portions, as well as primer binding sites or their sequence portions, may for example belong to sequence sections of a target sequence.

In one embodiment, the nucleic acid to be amplified corresponds to the target sequence.

In another embodiment, the target sequence forms part of the sequence of the nucleic acid sequence of the first amplification to be amplified. Such a target sequence may be flanked by 3 'side and / or 5' side of further sequences. These further sequences may include, for example, binding sites for primers or their moieties, and / or comprising primer sequences or their moieties, and / or binding sites for detection probes, and / or adapter sequences for complementary binding to a solid phase (eg, Im Frame of microarrays, or bead-based analyzes) and / or include barcoding sequences for a digital signature of sequences.

In order for the amplification to start, a nucleic acid chain must be added to the reaction mixture at the beginning of the reaction, which occurs as an initial template for the synthesis of the nucleic acid chain to be amplified. This nucleic acid chain is called the start nucleic acid chain. This start nucleic acid chain predetermines the arrangement of individual sequence elements which are important for the formation / synthesis / exponential amplification of a nucleic acid chain to be amplified.

In a preferred embodiment, the initial template (starting nucleic acid chain), which is fed to an amplification reaction at the beginning, or which is added to the reaction mixture, corresponds to the sequence composition of the nucleic acid chain to be amplified.

In initial stages of the amplification reaction and in its further course, the respective primers bind to the corresponding binding sites in the starting nucleic acid chain and initiate the synthesis of specific primer extension products. Such specific primer extension products accumulate exponentially during amplification and increasingly assume the role of templates for synthesis of complementary primer extension products in exponential amplification.

As a result of the repeated template-dependent synthesis processes in the context of an exponential amplification, the nucleic acid chain to be amplified is thus formed.

Towards the end of an amplification reaction, the major product of the reaction (the nucleic acid to be amplified) may be predominantly single-stranded or predominantly a complementary duplex. This can be determined, for example, by the relative concentrations of both primers and corresponding reaction conditions.

Equivalents of the nucleic acid to be amplified include nucleic acids having substantially identical information content. For example, complementary strands of a nucleic acid to be amplified have identical information content and can be said to be equivalent.

target sequence

A target sequence in one embodiment is a segment of a nucleic acid chain to be amplified which can serve as a characteristic sequence of the nucleic acid to be amplified. This target sequence can serve as a marker for the presence or absence of another nucleic acid. This other nucleic acid thus serves as the source of the target sequence and may for example be a genomic DNA or RNA or parts of genomic DNA or RNA (eg mRNA), or equivalents of genomic DNA or RNA Be RNA of an organism (eg cDNA, modified RNA such as rRNA, tRNA, microRNA, etc.), or comprise defined changes in the genomic DNA or RNA of an organism, for example mutations (eg deletions, insertions, substitutions, additions, sequence amplification, eg repeat propagation in the context of microsatellite instability), splice variants, rearrangement variants (eg T cell receptor variants), etc. The individual target sequences may represent a phenotypic trait, for example for antibiotic resistance or prognostic information and thus for diagnostic assays / Tests of interest. As a source / origin of a target sequence, such a nucleic acid may comprise, for example, the target sequence as a sequence element of its strand. A target sequence can thus serve as a characteristic marker for a particular sequence content of another nucleic acid.

The target sequence can be single-stranded or double-stranded. It may be substantially identical to the nucleic acid of the first amplification to be amplified or may be only part of the nucleic acid to be amplified.

Equivalents of the target sequence include nucleic acids with substantially identical information content. For example, complementary strands of a target sequence have identical informational content and can be said to be equivalent; RNA and DNA variants of a sequence are also examples of equivalent informational content.

As part of the material preparation prior to amplification reaction, such a target sequence can be isolated from its original sequence environment and prepared for the amplification reaction.

In a preferred embodiment, a nucleic acid to be amplified comprises a target sequence. In one embodiment, the target sequence corresponds to the nucleic acid to be amplified. In a further preferred embodiment, a starting nucleic acid chain comprises 1.1 a target sequence. In one embodiment, the target sequence corresponds to a starting nucleic acid chain 1.1

The target sequence may be fully or partially during the first amplification as part of the first amplification fragment 1.1 be included. In this case, for example, the second primer extension product comprises the target sequence or its portions and the second primer extension product thus comprises the strand complementary to this target sequence. This strand has an equivalent information content.

The target sequence may be fully or partially during the second amplification as part of the second amplification fragment 2.1 be included. For example, the fourth primer extension product comprises the target sequence or its portions, and the third primer extension product thus comprises the strand complementary to this target sequence. This strand has an equivalent information content.

Start-nucleic acid chain

In order for the first amplification to start, at the beginning of the reaction, a nucleic acid chain must be added to the reaction mixture, which acts as an initial template for the synthesis of the nucleic acid chain to be amplified ( 1 ). This nucleic acid chain is used as the starting nucleic acid chain of the first amplification (starting nucleic acid chain 1.1 ) designated. This start nucleic acid chain predetermines the arrangement of individual sequence elements which are important for the formation / synthesis / exponential amplification of a nucleic acid chain to be amplified.

Such a starting nucleic acid chain can be present in single-stranded or double-stranded form at the beginning of the reaction. When the complementary strands of the starting nucleic acid chain are separated, regardless of whether the nucleic acid was originally double- or single-stranded, the strands can serve as a template for the synthesis of specific complementary primer extension products.

The startup nucleic acid chain 1.1 in one embodiment comprises a target sequence.

A starting nucleic acid chain further comprises at least one predominantly single-stranded sequence segment, to which at least one of the primers of the amplification system can bind predominantly complementary with its 3 'segment, so that the polymerase used such a primer, when hybridized to the starting nucleic acid chain, matrizenspezifsiche under installation of dNTPs can extend.

In order for the second amplification to start, at the beginning of the second amplification reaction, a nucleic acid chain must be added to the reaction mixture, which serves as an initial template for the synthesis of the second amplification fragments occurs ( 1 ). This nucleic acid chain is used as the starting nucleic acid chain of the second amplification (starting nucleic acid chain 2.1 ) designated. This startup nucleic acid chain 2.1 specifies the arrangement of individual sequence elements which are important for the formation / synthesis / exponential amplification of a nucleic acid chain to be amplified.

This is where the first amplification fragment generated during the first amplification serves 1.1 (Also referred to as the nucleic acid chain to be amplified) as a starting nucleic acid chain 2.1 for the second amplification. This startup nucleic acid chain 2.1 thus comprises the first and second primer extension products generated in the first amplification. The startup nucleic acid chain 2.1 in one embodiment comprises a target sequence.

PCR amplification (also called second amplification)

PCR is a polymerase chain reaction which generally serves to amplify DNA fragments. Many variants of a PCR reaction are known in the art. Among other things, an allele-specific PCR, genotyping PCR, an asymmetric PCR, a solid-phase PCR, a digital PCR (partitioning / micro-PCR), multiplex PCR, real-time PCR using probes or intercalating dyes (eg Molecular beacons or 5'-3 'exonuclease or two oligonucleotide probes with FRET pairs), clamping PCR using blocking probes, quantitative PCR, nested PCR etc. A person skilled in the art will be referred to the numerous reviews which describe individual PCR procedures.

Generally, during PCR, an increase in nucleic acid fragments defined by the primer position occurs. In general, at least two temperature ranges are used: a low temperature temperature range for binding of primers to their predominantly specific sequence segments and extension to form a complementary strand and at least one temperature range involving predominantly sequence-unselective separation of Strands are made so that freshly synthesized primer extension products separate from their templates.

Many techniques have been developed to influence the course of PCR, e.g. Hotstart polymerases, hot-start oligonucleotides. Activatable primers (e.g., by 3'-5 'proofreading activity of polymerases).

PCR can be performed in liquid phase using PCR tubes / microtiter plates or on a solid phase or in microfluidic systems or in situ.

PCR primer pair (the third and the fourth primer oligonucleotide)

(Components of the second amplification)

Typically, includes two oligonucleotides capable of supporting amplification of a nucleic acid fragment by a PCR method.

In general, a PCR primer pair comprises a third and a fourth primer oligonucleotides. Such primers are known in the art.

For better clarity, PCR primers are used here as generally P3 and P4 (as opposed to a controller-independent amplification P1 and P2 ).

Many variants of PCR primers are known to a person skilled in the art. Typically, they are oligonucleotides of about 15 to about 60 nucleotides in length comprising at least in their 3 'segment sequences capable of binding to complementary segments of a target sequence and using this target sequence as a template to initiate template-dependent synthesis by means of a polymerase.

Many other functionalities, such as dye labeling (e.g., with FAM, TAMRA, Cy5), or quenchers (e.g., BHQ1), affinity tag (e.g., biotin, digoxigenin), or additional sequences, e.g. for sequence barcoding (use of specific tag sequences) or use of sequences for library construction are known to a person skilled in the art. PCR primers can be used in the solution or also coupled to a solid phase (e.g., beads, micrititer plates). These variants are also well known to a person skilled in the art.

Controller oligonucleotide:

The controller oligonucleotide ( 1 . C1.1 ) is a preferably single-stranded nucleic acid chain which includes a predefined substantially complementary sequence in a portion of the first primer extension product which is specifically generated as part of the amplification of the nucleic acid to be amplified (first amplification). This allows the controller oligonucleotide to bind substantially complementary to the first primer oligonucleotide and at least to the 5 'segment of the specific extension product of the first primer oligonucleotide. In one embodiment, the controller oligonucleotide includes nucleotide modifications in its inner sequence segment which prevent the polymerase from synthesizing a complementary strand using the controller oligonucleotide as a template when the first and / or third primer oligonucleotides are attached to the controller Oligonucleotide is bound complementarily. In one embodiment, it is advantageous if the controller oligonucleotide consists entirely of modified nucleotides that block its template function. In a further embodiment, it is advantageous if the controller oligonucleotide consists only in part of modified nucleotides which block its template function. The controller oligonucleotide is further capable of completely or partially displacing the second specific primer extension product from binding with the first specific primer extension product under strand reaction under the chosen reaction conditions. In this case, the controller oligonucleotide with its complementary regions is attached to the first specific primer extension product. Upon successful binding between the controller oligonucleotide and the first specific primer extension product, this results in the recovery of a single-stranded state of the 3'-terminal segment of the second specific primer extension product suitable for binding the first primer oligonucleotide, such that new primer extension reaction can take place. During synthesis of the second primer extension product, the controller oligonucleotide may be separated from binding with the first primer extension product by strand displacement, for example, by polymerase and / or by the second primer oligonucleotide.

Detektons system:

A detection system comprises at least one oligonucleotide probe and at least one fluorescent reporter (a fluorophore). The detection system should be capable of carrying out the synthesis of the first and / or the second and / or the third and / or the fourth primer extension product ( P1.1 -ext, P2.1 -ext, P3.1 -ext, P4.1 -Ext). This is done by using oligonucleotide probes, which are able to bind to the respective primer extension product and thereby generate a specific signal, or bring about a change in the signal. This change may be an increase or decrease in fluorescence intensity. A detection system may further comprise additional components. Such components preferably include fluorescence quencher and / or donor fluorophore. Furthermore, the detection system may also comprise the activator oligonucleotide. The array of fluorescent reporter, fluorescence quencher, donor phluorophore on the oligonucleotide probe or on the pair comprising oligonucleotide probe and activator oligonucleotide enables the binding events to the first primer extension product to be detected.

Fluorescence reporter - a fluorophore is a chemical compound or molecule that is able to emit electromagnetic radiation (light) when excited by electromagnetic radiation (emission). This radiation emitted by the fluorophore (emission) can be detected as a fluorescence signal by suitable technical means. Such a reporter can be covalently bound to an oligonucleotide. Many fluorophores are known which can be coupled to oligonucleotides (e.g., FAM, TAMRA, HEX, ROX, Cy dyes, Alexa dyes).

Fluorescence Quencher - A quencher is a chemical compound / molecule capable of reducing the emission of a fluorophore by direct contact (contact quenching) or by energy transfer (e.g., as FRET). Typically, a quencher must be placed in close proximity to the fluorophore for significant signal attenuation.

Distances are advantageous for a significant signal reduction of a fluorescence reporter by a quencher between the reporter and quencher of less than 25 nucleotides, more preferably less than 15 nucleotides, more preferably less than 5 nucleotides.

To overcome a signal attenuation of a fluorescent reporter by a quencher, the distance between these components must be increased accordingly, it is advantageous if the distance between the reporter and quencher of more than 15 nucleotides, more preferably more than 20 nucleotides, particularly advantageous to more is increased as 40 nucleotides. In this case, the distance caused by the nucleotide sequence must be due to an extended nucleotide sequence. For example, when forming hairpin structures, the spatial distance may no longer exist.

Especially with FRET-based quenchers, a sufficient spectral overlap between the emission spectrum of a fluorophore and the absorption spectrum of a quencher is advantageous. Therefore, fluorophore-quencher pairs are preferred which have more than 25% spectral overlap. (e.g., FAM / TAMRA).

Donor phluorophors - are chemical compounds / molecules that are capable of absorbing electromagnetic radiation and transferring it by energy transfer (eg, as FRET) to another fluorophore (acceptor) to stimulate that fluorophore, and as a result even light emission generated. Upon emission, a fluorescence signal is generated. As a rule, donor and acceptor form a fluorescence resonance energy transfer pair (a FRET pair). In general, a donor must be placed in close proximity to the acceptor (fluorophore) for this signal generation to occur to any significant extent.

Advantageous for signal generation of a fluorescent reporter due to FRET from a donor fluorophore are distances between the reporter and donor of less than 25 nucleotides, more preferably less than 15 nucleotides, more preferably less than 5 nucleotides.

The removal of the FRET effect from donor to acceptor is usually achieved by increasing the distance between both partners of a FRET pair. It is advantageous if the distance between the reporter and donor is increased from more than 15 nucleotides, better to more than 20 nucleotides, particularly advantageously more than 40 nucleotides.

Furthermore, a sufficient spectral overlap between the emission spectrum of a donor and the absorption spectrum of an acceptor is generally advantageous. Therefore, preference is given to FRET pairs having more than 25% in spectral overlap (e.g., FAM / Cy3).

According to the invention, the spatial distance between the individual components is preferably effected by connecting two components via a nucleotide sequence which can hybridize with a specific part of the target sequence.

Strand displacement (Strand Displacement):

This is a process which, by the action of a suitable agent for a complete or partial separation of a first double strand (consisting for example of A1 and B1 Strand) and for the simultaneous / parallel formation of a new second double strand, wherein at least one of the strands ( A1 or B1 ) are involved in the training of this new second strand. One can distinguish two forms of strand displacement here.

In a first form of strand displacement, the formation of a new second duplex may be accomplished using an already existing complementary strand which is generally in single-stranded form at the beginning of the reaction. In this case, the means of strand displacement (for example, a preformed single-stranded strand C1 which is a complementary sequence to the strand A1 has, on the first already formed double strand ( A1 and B1 ) and goes with the strand A1 a complementary bond, causing the strand B1 from the bond with the strand A1 is displaced. If the displacement of the B1 Completely expires, so is the result of C1 Action a new double strand ( A1: C1 ) and a single-stranded strand B1 , If the displacement of B1 incomplete, the result depends on several factors. For example, a complex of partially double-stranded A1: B1 and A1: C1 present as an intermediate.

In a second form of strand displacement, the formation of a new second duplex may occur under concurrent enzymatic synthesis of the complementary strand, with one strand of the first preformed duplex appearing as a template for synthesis by the polymerase. In this case, the means of strand displacement (for example, polymerase with a strand displacement ability) acts on the first already preformed double strand ( A1 and B1 ) and synthesizes one too strand A1 new complementary strand D1 , where at the same time the strand B1 from the bond with the strand A1 is displaced.

By the term "nucleic acid mediated strand displacement" is meant a sum / series of intermediate steps which may be in equilibrium with one another and, as a result, for the temporary or permanent opening of a first preformed duplex (consisting of complementary strands A1 and B1 ) and forming a new second duplex (consisting of complementary strands A1 and C1 ), in which A1 and C1 are complementary to each other.

It is known that an essential structural prerequisite for the initiation of strand displacement is the creation of a spatial proximity between a duplex end (preformed first duplex A1 and B1 ) and a single-stranded strand ( C1 ), which initiates strand displacement (where A1 and C1 can form a complementary strand). Such spatial proximity can preferably be achieved by means of a single-stranded overhang (in the literature examples with short overhangs are known, in English called "Toehold", see literature above), which the single-stranded strand ( C1 ) temporarily or permanently binds complementary, and thus complementary Segqmente the strand C1 and A1 brings in sufficient proximity, allowing a successful strand displacement of the strand B1 can be initiated. The efficiency of initiation of nucleic acid-mediated strand displacement is generally greater the closer the complementary segments of the strand are A1 and C1 be positioned to each other.

Another essential structural prerequisite for the efficient continuation of nucleic acid - mediated strand displacement in inner segments is the high complementarity between strands (eg between A1 and C1 ), which have to train a new double strand. For example, single nucleotide mutations (in C1 ) to interrupt strand displacement (described, for example, for branch migration).

The present invention makes use of the ability of complementary nucleic acids to sequence-dependent nucleic acid-mediated strand displacement.

Preferred embodiments of the invention are explained in more detail in the figures and examples.

Individual embodiments

Reaction conditions for first amplification

Reaction conditions include, but are not limited to, buffer conditions, temperature conditions, duration of reaction, and concentrations of respective reaction components.

In the course of the reaction, the amount of specific produced nucleic acid to be amplified accumulates in an exponential manner. The reaction comprising the synthesis of the extension products can be carried out for as long as necessary to produce the desired amount of the specific nucleic acid sequence. The process according to the invention is preferably carried out continuously. In a preferred embodiment, the amplification reaction proceeds at the same reaction temperature, wherein the temperature is preferably between 50 ° C and 70 ° C. In another embodiment, the reaction temperature can also be controlled variably, so that individual steps of the amplification run at respectively different temperatures.

The reagents required for the exponential amplification are preferably already present at the beginning of a reaction in the same batch. In another embodiment, reagents may also be added at later stages of the process.

Preferably, no helicases or recombinases are used in the reaction mixture to separate the newly synthesized duplexes of the nucleic acid to be amplified.

In a preferred embodiment, the reaction mixture does not include biochemical energy-donating compounds such as ATP.

The amount of nucleic acid to be amplified at the beginning of the reaction can be between a few copies and several billion copies in one run. For diagnostic applications, the amount of nucleic acid chain to be amplified may be unknown.

The reaction may also contain other nucleic acids which are not to be amplified. These nucleic acids can be derived from natural DNA or RNA or their equivalents. In one embodiment, control sequences are in the same approach, which should also be amplified parallel to the nucleic acid to be amplified.

Preferably, a molar excess of about 10 ³ : 1 to about 10 ¹⁵ : 1 (primer: template ratio) of the inserted primers and the controller oligonucleotide is added to the reaction mixture comprising template strands for the synthesis of the nucleic acid sequence to be amplified.

The amount of target nucleic acids may not be known if the method of the invention is used in diagnostic applications, so that the relative amount of the primer and the controller oligonucleotide relative to the complementary strand can not be determined with certainty. The amount of primer added will generally be present in molar excess relative to the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.

The concentrations of primer-1, primer-2 and controller oligonucleotides used are, for example, in the range between 0.01 μmol / l and 100 μmol / l, preferably between 0.1 μmol / l and 100 μmol / l, preferably between 0, 1 μmol / l and 50 μmol / l, more preferably between 0.1 μmol / l and 20 μmol / l. The high concentration of components can increase the rate of amplification. The respective concentrations of individual components can be varied independently of one another in order to achieve the desired reaction result.

The concentration of polymerase ranges between 0.001 μmol / l and 50 μmol / l, preferably between 0.01 μmol / l and 20 μmol / l, more preferably between 0.1 μmol / l and 10 μmol / l.

The concentration of individual dNTP substrates ranges between 10 .mu.mol / l and 10 mmol / l, preferably between 50 .mu.mol / l and 2 mmol / l, more preferably between 100 .mu.mol / l and 1 mmol / l. The concentration of dNTP can affect the concentration of divalent metal cations. If necessary, this will be adjusted accordingly.

As divalent metal cations, Mg2 +, for example, are used. As the corresponding anion, for example, Cl, acetate, sulfate, glutamate, etc. can be used. The concentration of divalent metal cations, for example, is adjusted to the optimum range for each polymerase and comprises ranges between 0.1 mmol / l and 50 mmol / l, more preferably between 0.5 mmol / l and 20 mmol / l, more preferably between 1 mmol / l and 15 mmol / l.

The enzymatic synthesis is generally carried out in a buffered aqueous solution. As buffer solutions, dissolved conventional buffer substances, such as Tris-HCl, Tris-acetate, potassium glutamate, HEPES buffer, sodium glutamate in conventional concentrations can be used. The pH of these solutions is usually between 7 and 9.5, preferably about 8 to 8.5. The buffer conditions can be adapted, for example, according to the recommendation of the manufacturer of the polymerase used.

Other substances, such as so-called Tm depressants (e.g., DMSO, betaines, TPAC), etc., can be added to the buffer. Such substances reduce the melting temperature ("Tm depressors") of double strands and thus can have a positive influence on the opening of double strands. Polymerase stabilizing components such as Tween 20 or Triton 100 can also be added in conventional amounts to the buffer. EDTA or EGTA can be added to complex heavy metals in conventional amounts. Polymerase stabilizing substances such as trehalose or PEG 6000 may also be added to the reaction mixture.

Preferably, the reaction mixture contains no inhibitors of the strand displacement reaction and no inhibitors of polymerase-dependent primer extension.

In one embodiment, the reaction mixture contains DNA-binding dyes, preferably intercalating dyes, e.g. EvaGreen or SybrGreen. Such dyes may optionally enable the detection of the formation of new nucleic acid chains.

The reaction mixture can furthermore contain proteins or other substances which, for example, originate from an original material and which preferably do not influence the amplification.

Preferred embodiments of Reaktionsbedinungen

The temperature has a significant influence on the stability of the double strands.

In a preferred embodiment, no temperature conditions are used during the amplification reaction, which essentially result in the separation of double strands of the nucleic acid to be amplified in the absence of controller oligonucleotide. This is to ensure that the double-stranded separation of nucleic acid chains to be amplified is dependent on the presence of the controller oligonucleotide throughout the course of the amplification.

At a temperature approximately equal to the measured melting temperature (Tm) of the nucleic acid to be amplified, a spontaneous separation of both strands of the nucleic acid to be amplified occurs, such that the influence of the controller oligonucleotide on separation of synthesized strands and thus on the sequence specificity of the amplification is minimal until it is limited.

For example, with exponential amplification that is less sequence specific (i.e., less controller oligonucleotide dependent), the reaction temperature may be around the melting temperature (i.e., Tm plus / minus 3 ° to 5 ° C) of the nucleic acid to be amplified. At such a temperature, sequence differences between the controller oligonucleotide and the synthesized primer extension product can generally be well tolerated in a strand displacement reaction.

Even in the temperature range from about (Tm minus 3 ° C) to about (Tm minus 10 ° C), there may still be a spontaneous strand separation of synthesized primer extension products, albeit with lower efficiency. The influence of controller oligonucleotide on the sequence specificity of the nucleic acid to be amplified is higher than at temperature conditions around the melting temperature (Tm) of the nucleic acid chain to be amplified.

Although the strand separation with decreasing reaction temperature takes place mainly due to interaction of the newly synthesized double strand with controller oligonucleotide, However, duplexes from primer at extension temperature under the conditions mentioned can also spontaneously decay, i. without sequence-dependent strand displacement by controller oligonucleotide. For example, the reaction temperature with a reduced sequence-specific amplification in ranges between about (Tm minus 3 ° C) and about (Tm minus 10 ° C), preferably between about (Tm minus 5 ° C) and about (Tm minus 10 ° C). At such temperature, sequence differences between controller oligonucleotide and the synthesized primer extension product are less tolerated in a strand displacement reaction.

A high sequence specificity of the amplification of the method is achieved, in particular, when the newly synthesized strands of the nucleic acid to be amplified can not spontaneously dissociate into single strands under reaction conditions. In such a case, the sequence-specific strand displacement by controller oligonucleotide plays a crucial role in sequence-specific strand separation and is significantly responsible for the sequence specificity of the amplification reaction. This can generally be achieved if the reaction temperature is well below the melting temperature of both strands of the nucleic acid to be amplified and no further components are used for strand separation, for example no helicases or recombinases. For example, the reaction temperature in a sequence-specific amplification in ranges between about (Tm minus 10 ° C) and about (Tm minus 50 ° C), preferably between about (Tm minus 15 ° C) and about (Tm minus 40 ° C), better between about (Tm minus 15 ° C) and about (Tm minus 30 ° C).

In a preferred embodiment of the amplification, the maximum reaction temperature during the entire amplification reaction is not increased above the melting temperature of the nucleic acid chain to be amplified.

In a further embodiment of the amplification, the reaction temperature can be increased at least once above the melting temperature of the nucleic acid chains to be amplified. The increase in temperature can be carried out, for example, at the beginning of the amplification reaction and lead to denaturation of double strands of a genomic DNA. It must be noted that during such Step, the dependence of the double-stranded separation is removed from the effect of the controller oligonucleotide, or at least significantly reduced.

The reaction temperatures of the individual steps of the amplification reaction can be in the range of about 15 ° C to about 85 ° C, more preferably in the range of about 15 ° C to about 75 ° C, preferably in the range of about 25 ° C to about 70 ° C.

In the examples 2 and 3 described below, a reaction temperature of the amplification reaction of 65 ° C was used, wherein the Tm of the nucleic acids to be amplified between about 75 ° C and about 80 ° C. Thus, the duplex of the nucleic acid to be amplified was stable under reaction conditions and the amplification reaction sequence-specific.

In general, the reaction temperature for each individual reaction step can be optimally adjusted, so that such a temperature is brought about for each reaction step. The amplification reaction thus comprises a repetitive change of temperatures, which are repeated cyclically. In an advantageous embodiment of the method, reaction conditions are standardized for a plurality of reaction steps, so that the number of temperature steps is less than the number of reaction steps. In such a preferred embodiment of the invention, at least one of the steps of amplification occurs at a reaction temperature which differs from the reaction temperature of other steps of the amplification. The reaction is thus not isothermal, but the reaction temperature is changed cyclically.

For example, during amplification at least two temperature ranges are used, which are brought about alternately (cyclic change of temperatures between individual temperature ranges). For example, in one embodiment, the lower temperature range includes temperatures between 25 ° C and 60 ° C, more preferably between 35 ° C and 60 ° C, preferably between 50 ° C and 60 ° C, and the upper temperature range includes, for example, temperatures between 60 ° C and 75 ° C, better between 60 ° C and 70 ° C.

In a further embodiment, the lower temperature range includes, for example, temperatures between 15 ° C and 50 ° C, more preferably between 25 ° C and 50 ° C, preferably between 30 ° C and 50 ° C and the upper temperature range includes, for example, temperatures between 50 ° C and 75 ° C, better between 50 ° C and 65 ° C.

In a further embodiment, the lower temperature range includes, for example, temperatures between 15 ° C and 40 ° C, more preferably between 25 ° C and 40 ° C, preferably between 30 ° C and 40 ° C and the upper temperature range includes, for example, temperatures between 40 ° C and 75 ° C, better between 40 ° C and 65 ° C.

The temperature can be kept constant in the respective range or changed as a temperature gradient (decreasing or rising).

Further explanations on temperature settings are detailed in the following sections of embodiments.

Any induced temperature can be maintained for a period of time, resulting in an incubation step. The reaction mixture may thus be incubated for a period of time during amplification at a selected temperature. This time may be different times for each incubation step and may be responsive to the progress of the particular reaction at a given temperature (e.g., primer extension or strand displacement, etc.). The time of an incubation step may comprise the following ranges: between 0.1 sec and 10,000 sec, more preferably between 0.1 sec and 1000 sec, preferably between 1 sec and 300 sec, more preferably between 1 sec and 100 sec.

By such a temperature change, individual reaction steps can preferably be carried out at a selected temperature. As a result, yields of a particular reaction step can be improved. Within a synthesis cycle, temperature change or temperature change between individual temperature ranges may be required several times. Synthesis cycle may thus include at least one temperature change. Such a temperature change can be performed routinely, for example, in a PCR device / thermocycler as a time program.

In one embodiment, an amplification method is preferred in which at least one of the steps comprising strand displacement and at least one of the steps comprising primer extension reactions take place simultaneously and in parallel and under the same reaction conditions. In such an embodiment, for example, a primer extension reaction of at least one primer oligonucleotide (eg, of the first primer oligonucleotide) may be carried out preferably at temperature conditions in the lower temperature range. On the other hand, the strand displacement with the assistance of controller oligonucleotide and the one further primer extension reaction (for example, of the second primer oligonucleotide) are preferably carried out in the reaction step in the upper temperature range.

In another embodiment, an amplification method is preferred in which at least one of the steps comprising strand displacement by the controller oligonucleotide and at least one of the steps comprising primer extension reaction are performed at different temperatures. For example, in such an embodiment, primer extension reactions of at least one primer oligonucleotide (e.g., the first primer oligonucleotide and / or the second primer oligonucleotide) may preferably be performed at lower temperature temperature conditions. By contrast, the strand displacement with the assistance of controller oligonucleotide preferably takes place in the reaction step in the upper temperature range.

In a further preferred embodiment, the steps of an amplification reaction proceed under identical reaction conditions.

In such an embodiment, the amplification process may be carried out under isothermal conditions, i. that no temperature changes are required to carry out the process. In such a preferred embodiment of the invention, the entire amplification reaction is carried out under constant temperature, i. the reaction is isothermal. The time of such a reaction includes, for example, the following ranges: between 100 sec and 30,000 sec, more preferably between 100 sec and 10,000 sec, even better between 100 sec and 1000 sec.

In the "Examples" section it is shown that it is possible to coordinate structures of individual reaction components and corresponding reaction steps to one another in such a way that an isothermal reaction is possible.

The sum of all process steps, which leads to a doubling of the amount of a nucleic acid chain to be amplified, can be referred to as a synthesis cycle. Such a cycle can be correspondingly isothermal or else characterized by changes in the temperature in its course. The temperature changes can be repeated from cycle to cycle and made identical.

Particularly advantageous are amplification processes in which the maximum achievable temperature essentially only permits strand separation with the assistance of controller oligonucleotide if more than 5 nucleotides of the third region of the controller oligonucleotide can form a complementary bond with the first primer extension product It is more advantageous if more than 10, more advantageously if more than 20 nucleotides of the controller oligonucleotide bind with the first primer extension product. In general, the longer the required binding between the controller oligonucleotide and the complementary strand of the first primer extension product, before the synthesized strands dissociate under reaction conditions, the more specific is the amplification reaction. Specifically, by extending or shortening the third portion of the controller oligonucleotide, the desired level of specificity can be determined.

A process step can be carried out at its constant temperature repetition over the entire duration of the process or at different temperatures.

Individual process steps can each be carried out in succession by adding individual components. In an advantageous embodiment, all reaction components necessary for the execution of an amplification are present at the beginning of an amplification in a reaction mixture.

The start of an amplification reaction can be carried out by addition of a component, for example by adding a nucleic acid chain comprising a target sequence (for example a starting nucleic acid chain), or a polymerase or divalent metal ions, or else by inducing reaction conditions, which are necessary for amplification, eg setting a required reaction temperature for one or more process steps.

The amplification can be carried out until the desired amount of nucleic acid to be amplified is reached. In another embodiment, the amplification reaction is carried out for a time which would have been sufficient in the presence of a nucleic acid to be amplified in order to obtain a sufficient amount. In another embodiment, the amplification reaction is carried out over a sufficient number of synthesis cycles (doubling times) which would have been sufficient in the presence of a nucleic acid to be amplified in order to obtain a sufficient amount.

The reaction can be stopped by various interventions. For example, by changing the temperature (e.g., cooling or heating, whereby, for example, polymerase is impaired in its function) or by adding a substance which stops a polymerase reaction, e.g. EDTA or formamide.

Following amplification, the amplified nucleic acid chain can be used for further analysis. In this case, synthesized nucleic acid chains can be analyzed by various detection methods. For example, fluorescently labeled oligonucleotide probes can be used, or sequencing methods (Sanger sequencing or next-generation sequencing), solid-phase analyzes such as microarray or bead-array analyzes, etc. The synthesized nucleic acid chain can be used as a substrate / template in further primer extension reactions become.

In an advantageous embodiment, the progress of the synthesis reaction is monitored during the reaction. This can be done, for example, by using intercalating dyes, e.g. Sybrgreen or Evagreen, or using labeled primers (e.g., Lux primers or Scorpion primers) or using fluorescently labeled oligonucleotide probes.

The detection of the change in fluorescence during amplification is implemented in a detection step of the method. The temperature and the duration of these steps can be adapted to the respective requirements of an oligonucleotide probe. The temperatures of the detection step include, for example, ranges between 20 ° C and 75 ° C, more preferably between 40 and 70 ° C, preferably between 55 and 70 ° C.

During the detection step, the reaction is illuminated with light of a wavelength which is capable of excitation into a used fluorophore of the detection system (a donor or a fluorescent reporter). The signal detection is usually parallel to excitation, whereby the specific fluorescence signal is detected and its intensity is quantified.

The amplification method can be used to verify the presence of a target nucleic acid chain in a biological material or diagnostic material as part of a diagnostic procedure.

Preferred embodiments for reaction conditions of the second amplification

The reaction conditions of a PCR are well known to a person skilled in the art.

Here, therefore, some preferred embodiments are exemplified.

• T1 = 55°C (+/- 5°C)
• T2 = 65°C (+/- 5°C)
• T3 = 95°C (+/- 5°C)
• T4 = 45°C (+/- 5°C)
• T5 = 70°C (+/- 5°C)

In one embodiment, the in 3 - 11 temperatures shown the following areas:

• T1 = 55 ° C (+/- 5 ° C)
• T2 = 65 ° C (+/- 5 ° C)
• T3 = 95 ° C (+/- 5 ° C)
• T4 = 45 ° C (+/- 5 ° C)
• T5 = 70 ° C (+/- 5 ° C)

• T1 = 55°C (+/- 10°C)
• T2 = 65°C (+/- 10°C)
• T3 = 95°C (+/- 10°C)
• T4 = 45°C (+/- 10°C)
• T5 = 70°C (+/- 10°C)

In a further embodiment, the in 3 - 11 temperatures shown the following areas:

• T1 = 55 ° C (+/- 10 ° C)
• T2 = 65 ° C (+/- 10 ° C)
• T3 = 95 ° C (+/- 10 ° C)
• T4 = 45 ° C (+/- 10 ° C)
• T5 = 70 ° C (+/- 10 ° C)

• T1 = 55°C (+/- 15°C)
• T2 = 65°C (+/- 10°C)
• T3 = 95°C (+/- 10°C)
• T4 = 45°C (+/- 20°C)
• T5 = 70°C (+/- 10°C)

In a further embodiment, the in 3 - 11 temperatures shown the following areas:

• T1 = 55 ° C (+/- 15 ° C)
• T2 = 65 ° C (+/- 10 ° C)
• T3 = 95 ° C (+/- 10 ° C)
• T4 = 45 ° C (+/- 20 ° C)
• T5 = 70 ° C (+/- 10 ° C)

• T1 = 55°C (+/- 5°C)
• T2 = 65°C (+/- 3°C)
• T3 = 95°C (+/- 10°C)
• T4 = 45°C (+/- 5°C)
• T5 = 70°C (+/- 3°C)

In a further embodiment, the in 3 - 11 temperatures shown the following areas:

• T1 = 55 ° C (+/- 5 ° C)
• T2 = 65 ° C (+/- 3 ° C)
• T3 = 95 ° C (+/- 10 ° C)
• T4 = 45 ° C (+/- 5 ° C)
• T5 = 70 ° C (+/- 3 ° C)

The time of incubation during individual temperature steps may in some embodiments be between 0.1 sec and 60 min, more preferably between 1 sec and 10 min, even better between 1 sec and 5 min.

The cyclic temperature changes are often referred to as PCR cycles. For example, a PCR cycle involves a complete change in the temperature of T1 . T2 on T3 ,

The temperature ranges used are primarily intended to support the following steps of the second amplification:

T1 . T2 . T4 and T5 Temperatures: hybridization of the third and fourth primers to their complementary segments in respective template strands and extension of a third and fourth primer extension product.

T3 Temperature is used to separate double strands comprising at least one of the primer extension products. All double-stranded nucleic acid chains (eg double-stranded target sequences, first amplification fragments) are considered 1.1 , second amplification fragments 2.1 , as well as intermediates P1.1 -Ext and P4.1 -Ext part 1 or P2.1 -Ext and P3.1 -Ext part 1) converted by the action of temperature in single-stranded form.

Furthermore, can T3 Temperature for activation of hot-start forms of the second polymerases are used (eg, which were inactivated by an antibody) or inactivation of a first polymerase. Furthermore, thermolabile controller oligonucleotides can be replaced by this temperature ( T3 ) are split. Furthermore, thermoactivatable primers three and four can be activated.

Thus, various temperatures can be used for the reaction control.

Furthermore, the choice of reaction temperatures in combination with respective PCR primers (the third and the fourth primer of the second amplification) can influence the production of products / formation of intermediates during the second amplification.

The amount of yields of individual products and intermediates depends on several factors. For example, higher concentrations of individual primers generally favor the yields of products / intermediates. Furthermore, the formation of products / intermediates can be influenced by reaction temperature and binding affinity of individual reactants to each other (affinity of binding of individual primers to their complementary primer binding sites within template strands): in general, for example, longer oligonucleotides bind better at higher temperatures than shorter oligonucleotides Furthermore, CG content may play a role in complementary sequence segments: CG-richer sequences also bind more strongly at higher temperatures than AT-rich sequences. Furthermore, modifications such as MGB or 2-amino-dA or LNA can increase the binding strength of primers to their respective complementary segments, which also results in preferential binding of primers at higher temperatures.

For example, longer primers are used for the PCR (eg between 25 and 60 nucleotides, with a CG content of over 40%), which with their complementary regions have a relatively higher Tm (eg from 60 to 70 ° C). This allows hybridization steps and primer extension steps of the second amplification to be carried out at higher temperatures (eg T2 or T5 ). Thus, in combination with relatively short first and second primers (from the first amplification), the reaction can be controlled to include primer extension products P4.1 -Ext and P3.1 Be primarily formed and the co-amplification of primer extension products starting from P1.1 and P2.1 can be largely suppressed during the PCR amplification.

For example, using third and fourth primers, including, for example, only a short 3 'segment that can bind to the target sequence or its equivalents (eg, this segment is between 6 and 15 nucleotides in length), it may be advantageous to first do a few cycles with a low temperature ( 10 A and B, A2.1 Phase). A similar institution also relates to PCR primers which, for example, mismatches to each only substantially complementary first amplification fragment 1.1 include.

During this phase with relatively low temperatures ( T1 and T4 ), a primer binding of the third and / or fourth primer to its complementary segments of the first amplification fragment, so that in spite of a small length of the complementary segment and / or existing mismatches, the polymerase hybridized starting from these primers to individual strands of the first amplification Fragment can perform a template-dependent primer extension and primer extension products comprising a third and a fourth primer oligonucleotide can be synthesized.

These PCR cycles are repeated at least twice, so that complementary strands can be synthesized. Thus, when repeating the cycles, the third and fourth primers themselves are also copied as templates, resulting in the formation of complete primer binding sites for the third and / or fourth primer oligonucleotide.

Subsequently, an increase of the temperature can take place ( A2.2 Phase) so that the third and fourth primers can bind to fully formed primer binding sites at higher temperature.

Use of higher temperatures ( T2 and T5 ) is advantageous, for example, in a homogeneous assay (when both amplification systems are present in the same reaction mixture). Thus, for example, the influence of the binding of the controller oligonucleotides to complementary regions of the target sequence of the third primer extension product ( P3.1 -Ext or P3.1 -Ext part) can be reduced.

In the second amplification, the enzymatic synthesis is generally carried out in a buffered aqueous solution. As buffer solutions, dissolved conventional buffer substances, such as Tris-HCl, Tris-acetate, potassium glutamate, HEPES buffer, sodium glutamate in conventional concentrations can be used. The pH of these solutions is usually between 7 and 9.5, preferably about 8 to 8.5. The buffer conditions can be adapted, for example, according to the recommendation of the manufacturer of the polymerase used.

Other substances, such as so-called Tm depressors (eg DMSO, betaines, TPAC) etc. can be added to the buffer. Such substances reduce the melting temperature ("Tm depressors") of double strands and thus can have a positive influence on the opening of double strands. Polymerase stabilizing components such as Tween 20 or Triton 100 can also be added in conventional amounts to the buffer. EDTA or EGTA may cause complexation of heavy metals in conventional Quantities are added. Polymerase stabilizing substances such as trehalose or PEG 6000 may also be added to the reaction mixture.

Preferred embodiments of a starting nucleic acid chain: for first amplification

The nucleic acid chain used or to be used at the beginning of an amplification reaction can be referred to as the starting nucleic acid chain ( 1 ).

Their function can be seen as representing the initial template that allows for correct positioning of primers, the synthesis sections between both primers, and the initiation of binding and extension procedures. In a preferred embodiment, a starting nucleic acid chain comprises a target sequence.

By binding of primers to their corresponding primer binding sites (PBS 1 and PBS 2 ) and initiation of appropriate primer extension reactions by generation of first primer extension products. These are synthesized as specific copies of the nucleic acid chain present at the beginning of the reaction.

In one embodiment, this nucleic acid chain (starting nucleic acid chain) to be used in the reaction mixture before the beginning of the amplification reaction can be identical to the nucleic acid chain to be amplified. The amplification reaction only increases the amount of such a nucleic acid chain.

In a further embodiment, the nucleic acid to be amplified and the starting nucleic acid chain differ in that the starting nucleic acid chain prescribes the arrangement of individual sequence elements of the nucleic acid chain to be amplified, but the sequence composition of the starting nucleic acid chain can deviate from the sequence of the nucleic acid chain to be amplified. For example, in the context of primer binding and extension during amplification, new sequence contents (based on starting nucleic acid chain) can be integrated into the nucleic acid chain to be amplified. Furthermore, sequence elements of a nucleic acid chain to be amplified may differ from such sequence elements of a starting nucleic acid chain in their sequence composition (e.g., primer binding sites or primer sequences). The starting nucleic acid serves only as an initial template for the specific synthesis of the nucleic acid chain to be amplified. This initial template may remain in the reaction mixture until the end of the amplification. Due to the exponential nature of the amplification, however, the amount of nucleic acid chain to be amplified at the end of an amplification reaction outweighs the amount of a starting nucleic acid chain added to the reaction.

In a further embodiment, the starting nucleic acid chain may comprise at least one sequence segment which is not amplified. Such a start nucleic acid chain is thus not identical to the sequence to be amplified. Such sections which are not to be amplified may, for example as a consequence of sequence preparation steps or as a consequence of preceding sequence manipulation steps, represent a sequence section of a starting nucleic acid chain.

In a preferred embodiment, the starting nucleic acid sequence to be inserted into the reaction mixture prior to initiation of the reaction includes at least one target sequence.

In a further embodiment, such a start nucleic acid chain includes at least one target sequence and still further sequences which are not non-target sequences. During amplification, sequence segments comprising the target sequence are multiplied exponentially and other sequence segments are either not or only partially exponentially propagated.

Construction of a starting nucleic acid chain (for first amplification)

An example of such a start nucleic acid chain is a nucleic acid sequence which includes a target sequence and which comprises a sequence fragment A and comprises a sequence fragment-B.

The sequence fragment A of the starting nucleic acid chain comprises a sequence which has significant homology with the sequence of one of the two primers used in the amplification or is substantially identical to the copiable portion of the 3 'segment of the one primer. In the synthesis of a complementary strand to this segment, a complementary sequence is generated which represents a corresponding primer binding site.

The sequence fragment B of the starting nucleic acid chain comprises a sequence which is suitable for complementarily binding a corresponding further primer or its 3 'segment to form an extension-capable primer-template complex, wherein sequence fragment A and sequence fragment B are relative to one another predominantly / preferably are not complementary.

In a preferred embodiment, the reaction mixture of an amplification process is given a starting nucleic acid chain which has the following properties:

Sequence fragment-A is preferably located in the 5 'segment of the starting nucleic acid chain. Preferably, this sequence fragment-A forms a boundary of the nucleic acid chain strand in the 5 'direction.

Preferably sequence fragment-B is downstream of sequence fragment-A.

In a preferred embodiment, this sequence fragment-B forms a boundary of the nucleic acid chain strand in the 3 'direction. In a further embodiment, this sequence fragment-B does not represent a limitation of the nucleic acid chain strand in the 3 'direction, but is flanked by further sequences from the 3' side. Preferably, these sequences are not targeting sequences and do not participate in exponential amplification.

In one embodiment, the target sequence comprises at least one of both Sequence Fragment A or Sequence Fragment B. In another embodiment, the target sequence is between sequence fragment A and sequence fragment B.

In one embodiment, 57 ), such a starting nucleic acid chain can serve as a template for the synthesis of a first primer extension product. The starting nucleic acid chain can be provided, for example, in the context of a primer extension reaction using the second primer oligonucleotide as a sequence segment of a longer starting nucleic acid chain during a preparatory step before exponential amplification and converted into a single-stranded form. The starting nucleic acid chain may be, for example, a genomic DNA or RNA and serves as a source of the target sequence. The start nucleic acid chain comprises a segment in the 5 'segment of the nucleic acid chain 1 , which comprises sequence sections of the second primer oligonucleotide and represents 5'-directional restriction, a segment 2 which includes a target sequence or its parts, a segment 3 which comprises a primer binding site for the first primer oligonucleotide and a segment 4 which locates a non-target sequence in the 3 'segment of the starting nucleic acid chain and thus the segment 3 flanked on the 3 'side.

In the context of the initiation of the amplification, the first primer, at least with the 3 'segment of its first region, can be linked to such a starting nucleic acid chain in the segment 3 and be extended accordingly in the presence of a polymerase and nucleotides. This results in a first primer extension product which is complementary to the template strand of the starting nucleic acid chain and is limited in its length. In the context of the amplification reaction, such a primer extension product can be detached from its template strand sequence-specific via the controller oligonucleotide so that corresponding sequence segments in the 3'-segment of the now synthesized first primer extension product are available as a binding site for the second primer oligonucleotide.

• • Sequenzsegment-1 (Segment 1 bezeichnet), welches eine Sequenz umfasst, die mit der Sequenz des zweiten Primers eine signifikante Homologie aufweist oder im Wesentlichen mit dem kopierbaren Anteil des 3'-Segments des zweiten Primers identisch ist. Dieses Sequenzsegment-1 liegt im 5'-Segment der Start-Nukleinsäurekette, vorzugsweise bildet dieses Sequenzsegment-1 eine Begrenzung des Nukleinsäurekette-Stranges in 5'-Richtung.
• • Das Sequenzsegment-3 (Segment 3 bezeichnet), welches eine Sequenz umfasst, welche zu komplementären Bindung des ersten Bereichs des ersten Primers oder seines 3'-Segmentes unter Ausbildung eines extensionsfähigen Primer-Matrizen-Komplexes geeignet ist. Vorzugsweise liegt das Sequenzsegment-3 stromabwärts vom Sequenzsegment-1.
• • Zielsequenz , welche teilweise oder ganz zwischen Segment 1 und Segment 3 liegt (dieser Anteil der Zielsequenz wird als Segment 2 bezeichnet). In einer bevorzugten Ausführungsform umfasst die Zielsequenz mindestens eines von Segment 1 und / oder Segment 3.
• • Optional umfasst die Start-Nukleinsäurekette im 3'-Segment flankierende Sequenzabschnitte (als Segment 4 bezeichnet), welche nicht amplifiziert werden.

In a preferred embodiment, a starting nucleic acid chain thus comprises the following sequence fragments ( 57 ):

• • sequence segment 1 (Segment 1 ) which comprises a sequence which has significant homology with the sequence of the second primer or is substantially identical to the copiable portion of the 3 'segment of the second primer. This sequence segment 1 lies in the 5 'segment of the starting nucleic acid chain, this sequence segment preferably forms 1 a limitation of the nucleic acid chain strand in the 5 'direction.
• • The sequence segment 3 (Segment 3 ) which comprises a sequence which is suitable for complementary binding of the first region of the first primer or its 3 'segment to form an extendible primer-template complex. Preferably, the sequence segment 3 downstream of the sequence segment 1 ,
• • Target sequence, which partially or completely between segment 1 and segment 3 (this portion of the target sequence is called a segment 2 designated). In a preferred embodiment, the target sequence comprises at least one segment 1 and / or segment 3 ,
• Optionally, the start nucleic acid chain in the 3 'segment comprises flanking sequence segments (as a segment 4 designated) which are not amplified.

In another embodiment, such a starting nucleic acid chain can serve as a template for the synthesis of a second primer extension product ( 58 ).

The starting nucleic acid chain can be provided, for example, in the context of a primer extension reaction using the first primer oligonucleotide as a sequence segment of a longer starting nucleic acid chain during a preparatory step before the exponential amplification and converted into a single-stranded form. The starting nucleic acid chain may be, for example, a genomic DNA or RNA and serves as the source of the target sequence (shown schematically as a double strand). The start nucleic acid chain comprises a segment in the 5 'segment of the nucleic acid chain 5 , which comprises sequence sections of the first primer oligonucleotide and represents 5'-directional restriction, a segment 6 which includes a target sequence or its parts, a segment 7 which comprises a primer binding site for the second primer oligonucleotide and a segment 8th which locates a non-target sequence in the 3 'segment of the starting nucleic acid chain and thus the segment 7 flanked on the 3 'side.

In the context of the initiation of the amplification, the second primer, at least with the 3 'segment of its first region, can be attached to such a starting nucleic acid chain in the segment 7 and extended in the presence of a polymerase and nucleotides correspondingly up to the stop portion of the first primer oligonucleotide. This results in a second primer extension product which is complementary to the template strand of the starting nucleic acid chain and is limited in its length. In the context of the amplification reaction, such a primer extension product can be detached from its template strand sequence-specific via the controller oligonucleotide so that corresponding sequence segments in the 3 'segment of the now synthesized second primer extension product are available as a binding site for the first primer oligonucleotide.

• • Sequenzsegment-5 (genannt Segment 5), welches eine Sequenz umfasst, die mit der Sequenz des ersten Bereichs des ersten Primers eine signifikante Homologie aufweist oder im Wesentlichen mit dem kopierbaren Anteil des ersten Bereichs des ersten Primers identisch ist. Dieses Segment-7 liegt im 5'-Segment der Start-Nukleinsäurekette und ist von einem nicht kopierbaren Oligonukleotid-Schwanz flankiert (analog zum ersten Primer-Oligonukleotid), vorzugsweise bildet dieses Segment - 7 eine Begrenzung des kopierbaren Nukleinsäurekette-Stranges in 5'-Richtung.
• • Das Sequenzfragment-7 (genannt Segment 7), welches eine Sequenz umfasst, welche zur komplementären Bindung eines zweiten Primers oder seines 3'-Segmentes unter Ausbildung eines extensionsfähigen Primer-Matrizen-Komplexes geeignet ist. Vorzugsweise liegt das Segment 7 stromabwärts vom Segment 5.
• • Zielsequenz , welche teilweise oder ganz zwischen Segment 5 und Segment 7 liegt (dieser Anteil der Zielsequenz wird als Segment 6 bezeichnet). In einer bevorzugten Ausführungsform umfasst die Zielsequenz mindestens eines von Segment 5 und / oder Segment 7.
• • Optional umfasst die Start-Nukleinsäurekette im 3'-Segment flankierende Sequenzabschnitte (als Segment 8 bezeichnet), welche nicht amplifiziert werden.

In this preferred embodiment, a start nucleic acid sequence comprises the following sequence fragments ( 58 ):

• • sequence segment 5 (called segment 5 ) comprising a sequence having significant homology with the sequence of the first region of the first primer or being substantially identical to the copiable portion of the first region of the first primer. This segment 7 lies in the 5 'segment of the starting nucleic acid chain and is flanked by a non-copyable oligonucleotide tail (analogous to the first primer oligonucleotide), preferably this segment - 7 forms a boundary of the copiable nucleic acid chain strand in the 5' direction.
• • the sequence fragment 7 (called segment 7 ), which comprises a sequence which is suitable for the complementary binding of a second primer or its 3 'segment to form an extension-capable primer-template complex. Preferably, the segment lies 7 downstream from the segment 5 ,
• • Target sequence, which partially or completely between segment 5 and segment 7 (this portion of the target sequence is called a segment 6 designated). In a preferred embodiment, the target sequence comprises at least one segment 5 and / or segment 7 ,
• Optionally, the start nucleic acid chain in the 3 'segment comprises flanking sequence segments (as a segment 8th designated) which are not amplified.

Function of the starting nucleic acid chain for the first amplification reaction

At the beginning of the amplification reaction, the starting nucleic acid chain serves as a template for initial formation of respective primer extension products. It thus represents the starting template for the nucleic acid chain to be amplified. The starting nucleic acid chain does not necessarily have to be identical to the nucleic acid chain to be amplified. By binding and extending both primers during the amplification reaction, essentially the two primers determine which sequences are generated at both terminal segments of the nucleic acid chain to be amplified as part of the amplification.

In a preferred embodiment of the method, non-denaturing reaction conditions are maintained for a double strand during the exponential amplification process. Therefore, it is advantageous if the starting nucleic acid chain has a restriction in its 5 'sequence segment which can be extended by a polymerase, resulting in a stop in the enzymatic extension of a corresponding primer. This limits the length of the primer extension fragments generated under reaction conditions. This can have an advantageous effect on strand displacement by controller oligonucleotide and lead to a dissociation of the respective strand, so that primer binding sites are converted into single-stranded state and thus become available for a re-binding of primers.

Preferred Embodiments of the First Primer Oligonucleotide (Primer-1)

• • Einen ersten Primer-Bereich im 3'-Segment des ersten Primer-Oligonukleotides, der an einen Strang einer zu amplifizierenden Nukleinsäurekette im Wesentlichen sequenzspezifisch binden kann
• • Einen zweiten Bereich, gekoppelt direkt oder via einen Linker an das 5'-Ende des ersten Primer-Bereichs des ersten Primer-Oligonukleotides, welcher einen Polynukleotid-Schwanz umfasst, welcher zur Bindung eines Controller-Oligonukleotids und Unterstützung der Strangverdrängung (Schritt c) durch Controller-Oligonukleotids geeignet ist, wobei der Polynukleotid-Schwanz unter Reaktionsbedingungen im wesentlichen einzelsträngig bleibt, d.h. keine stabile Hairpin-Struktur oder ds-Strukturen ausbildet, und vorzugsweise nicht von Polymerase kopiert wird.

The first primer oligonucleotide (primer 1 ) is a nucleic acid chain which includes at least the following ranges ( 14 and 15 ):

• A first primer region in the 3 'segment of the first primer oligonucleotide that is capable of substantially sequence-specific binding to a strand of a nucleic acid sequence to be amplified
• A second region, coupled directly or via a linker, to the 5 'end of the first primer region of the first primer oligonucleotide comprising a polynucleotide tail capable of binding a controller oligonucleotide and promoting strand displacement (step c). by controller oligonucleotide, wherein the polynucleotide tail remains substantially single-stranded under reaction conditions, ie, does not form a stable hairpin or ds structure, and is preferably not copied by polymerase.

The total length of the first primer oligonucleotide is between 10 and 80, preferably between 15 and 50, more preferably between 20 and 30 nucleotides or their equivalents (e.g., nucleotide modifications). The structure of the first primer oligonucleotide is adapted to undergo reversible binding to controller oligonucleotide under selected reaction conditions. Furthermore, the structure of the first primer oligonucleotide is adapted to its primer function. Furthermore, the structure is adapted so that a strand displacement can be performed by means of controller oligonucleotide. Overall, structures of the first and second regions are matched to each other so that exponential amplification can be performed.

In an advantageous embodiment of the invention, the first and the second region of the primer are coupled in a conventional 5'-3 'arrangement. In a further embodiment of the invention, the coupling of both sections takes place via a 5'-5 'bond, so that the second region has a reverse direction than the first region.

The coupling of areas between each other / each other is preferably covalent. In one embodiment, the coupling between the first and second regions is a 5'-3 'phosphodiester coupling conventional for DNA. In another embodiment, it is a 5'-5'-phosphodiester coupling. In a further embodiment, it is a 5'-3'-phosphodiester coupling, wherein between adjacent terminal nucleotides or nucleotide modifications of the two regions at least one linker (eg C3 . C6 . C12 or an HEG linker or an abasic modification).

Individual regions may include different nucleotide modifications. In this case, individual elements of nucleotides can be modified: nucleobase and backbone (sugar content and / or phosphate content). Furthermore, modifications may be used which lack or are modified at least one component of the standard nucleotide building blocks, e.g. PNA.

In a further embodiment, a second region of the first primer oligonucleotide comprises further sequences that do not bind to the controller oligonucleotide.

These sequences can be used for other purposes, e.g. to bond to the solid phase. These sequences are preferably located at the 5 'end of the polynucleotide tail.

In a further embodiment, a first primer oligonucleotide may comprise characteristic label. Examples of such a label are dyes (eg FAM, TAMRA, Cy3, Alexa 488 etc.) or biotin or other groups which can be specifically bound, eg digoxigenin.

The first primer region of the first primer oligonucleotide

The sequence length is between about 3-30 nucleotides, preferably between 5 and 20 nucleotides, the sequence being predominantly complementary to the 3 'segment of a strand of the nucleic acid chain to be amplified. In particular, this primer region must be able to specifically bind to the complementary 3 'segment of a second primer extension product. This first area should be copied in reverse synthesis and also serves as a template for 2nd strand. The nucleotide building blocks are preferably linked to one another via customary 5'-3 'phosphodiester bond or phosphothioester bond.

• • natürliche Nukleotide (dA, dT, dC, dG etc.) oder deren Modifikationen ohne veränderte Basen-Paarung
• • Modifizierte Nukleotide, 2-Amino-dA, 2-thio-dT oder andere Nukleotid-Modifikationen mit abweichender Basen-Paarung.

The first primer region preferably includes nucleotide monomers which do not or only insignificantly affect the function of the polymerase, for example:

• • natural nucleotides (dA, dT, dC, dG etc.) or their modifications without altered base pairing
• • Modified nucleotides, 2-amino-dA, 2-thio-dT or other nucleotide modifications with deviant base pairing.

In a preferred embodiment, the 3'-OH end of this region is preferably free of modification and has a functional 3'-OH group which can be recognized by polymerase. The first primer region serves as the initiator of the synthesis of the first primer extension product in the amplification. In a further preferred embodiment, the first region comprises at least one phosphorothioate compound so that no degradation of 3'-end of the primers by 3'-exonuclease activity of polymerases can take place.

The sequence of the first region of the first primer oligonucleotide and the sequence of the second region of the controller oligonucleotide are preferably complementary to one another.

In one embodiment, the first primer region or its 3 'segment can bind to sequence segments of a target sequence.

The second region of the first primer oligonucleotide

The second region of the first primer oligonucleotide is preferably a nucleic acid sequence comprising at least one polynucleotide tail which preferably remains uncopied during the synthesis reaction of polymerase and which is capable of binding to the first region of the controller oligonucleotide. The segment of the second region, which predominantly undergoes this binding with the controller oligonucleotide, may be referred to as the polynucleotide tail.

Furthermore, the second region of the first primer oligonucleotide must not only specifically bind the controller oligonucleotide under reaction conditions, but also participate in the process of strand displacement by means of controller oligonucleotide. The structure of the second region must therefore be suitable for bringing about spatial proximity between the controller oligonucleotide and the corresponding duplex end (in particular, the 3 'end of the second primer extension product).

The design of the structure of the second region of the first primer oligonucleotide is shown in more detail in several embodiments. It takes into account the arrangement of the oligonucleotide segments and modifications used, which lead to a stop in the polymerase-catalyzed synthesis.

The length of the second region is between 3 and 60, preferably between 5 and 40, preferably between 6 and 15 nucleotides or their equivalents.

The sequence of the second area may be chosen arbitrarily. Preferably, it is not complementary with the nucleic acid to be amplified and / or with the second primer oligonucleotide and / or with the first region of the first primer oligonucleotide. Furthermore, it preferably contains no self-complementary segments, such as hairpins or stemmloops.

The sequence of the second region is preferably matched to the sequence of the first region of the controller oligonucleotide, so that both sequences can bind under reaction conditions. In a preferred embodiment, this bond is reversible under reaction conditions: there is thus a balance between components bound together and unbound components.

The sequence of the second region of the first primer oligonucleotide is preferably chosen such that the number of complementary bases that can bind to the first region of the controller oligonucleotide is between 1 and 40, more preferably between 3 and 20, preferably between 6 and 15 lies.

The function of the second region consists inter alia in binding of the controller oligonucleotide. In one embodiment, this binding is preferably specific so that a second region of a first primer oligonucleotide can bind a specific controller oligonucleotide. In another embodiment, a second region may bind more than one controller oligonucleotide under reaction conditions.

There is generally no need for a perfect match in sequence between the second region of the first primer oligonucleotide and the first region of the controller oligonucleotide. The degree of complementarity between the second region of the first primer oligonucleotide and the first region of the controller oligonucleotide may be between 20% and 100%, more preferably between 50% and 100%, preferably between 80% and 100%. The respective complementary regions may be positioned immediately adjacent to each other or may comprise non-complementary sequence segments therebetween.

The second region of the first primer oligonucleotide, in one embodiment, may include at least one Tm modifying modification. By introducing such modifications, the stability of the binding between the second region of the first primer oligonucleotide and the first region of the controller oligonucleotide can be modified. For example, Tm enhancing modifications (nucleotide modifications or non-nucleotide modifications) can be used, such as LNA nucleotides, 2-amino adenosines, or MGB modifications. On the other hand, Tm-lowering modifications may also be used, such as inosine nucleotide. In the structure of the second area also linkers (eg C3 . C6 , HEG linkers) are integrated.

For strand displacement, the controller oligonucleotide must be placed in close proximity to the double-stranded end of the nucleic acid to be amplified. This double-stranded end consists of segments of the first primer region of the first primer extension product and a correspondingly complementary thereto 3 'segment of the second primer extension product.

The polynucleotide tail predominantly complements the controller oligonucleotide under reaction conditions, thereby causing a transient approach of the second region of the controller oligonucleotide and the first region of an extended primer extension product such that a complementary bond between these elements is initiated as part of a strand displacement process can.

In one embodiment, binding of the controller oligonucleotide to the polynucleotide tail of the first primer oligonucleotide directly results in such contact. This means that the polynucleotide tail and the first primer region of the first primer oligonucleotide must be directly coupled to each other. By virtue of such an arrangement, upon binding of a controller oligonucleotide in its first region, direct contact may be made between complementary bases of the second region of the controller oligonucleotide and corresponding bases of the first primer region, such that strand displacement may be initiated.

In another embodiment, structures of the second region of the first primer oligonucleotide are located between structures of the polynucleotide tail and the first primer region. After binding of a controller oligonucleotide to the polynucleotide tail, it is thus not positioned directly at the first primer region, but at a certain distance from it. The structures between the uncopiable polynucleotide tail and the copiable first primer region of the primer oligonucleotide can generate such a spacing. This distance has a value which is between 0.1 and 20 nm, preferably between 0.1 and 5 nm, more preferably between 0.1 and 1 nm.

Such structures represent, for example, linkers (eg C3 or C6 or HEG linker) or segments that are not complementary to controller oligonucleotide (eg, in the form of non-complementary, non-copyable nucleotide modifications). The length of these structures can generally be measured in chain atoms. This length is between 1 and 200 atoms, preferably between 1 and 50 chain atoms, preferably between 1 and 10 chain atoms.

In order for the polynucleotide tail of polymerase to remain uncopyable under amplification conditions, the second region of the first primer oligonucleotide generally comprises sequence structures that cause the polymerase to stop in the synthesis of the second primer extension product after the polymerase releases the polymerase first primer area has successfully copied. These structures are said to prevent copying of the polynucleotide tail of the second region. The polynucleotide tail thus preferably remains uncoupled from the polymerase.

In one embodiment, such structures are between the first primer region and the polynucleotide tail.

In another embodiment, the sequence of the polynucleotide tail may include nucleotide modifications that result in the termination of the polymerase. Thus, a sequence segment of the second region of the first primer oligonucleotide may comprise both functions: it is both a polynucleotide tail and a polymerase-terminating sequence of nucleotide modifications.

Modifications in the second region of the first primer oligonucleotide which result in a synthesis stop and thus leave the polynucleotide tail uncopyable are summarized in this application by the term "first blocking moiety or a stop region".

In the following, further embodiments of structures are given, which can lead to a stop in the synthesis of the second strand.

Several building blocks in oligonucleotide synthesis are known which prevent the polymerase from reading the template and lead to the termination of polymerase synthesis. For example, non-copyable nucleotide modifications or non-nucleotide modifications are known. There are also synthesis types / arrangements of nucleotide monomers within an oligonucleotide which result in the termination of the polymerase (e.g., 5'-5 or 3'-3 'arrangement). Primer oligonucleotides with a non-copyable polynucleotide tail are also known in the art (e.g., Scorpion primer structures). Both primer variants describe primer oligonucleotide structures which are able to initiate the synthesis of a strand on the one hand so that a primer extension reaction can take place. The result is a first strand, which also integrates the primer structure with tail in the primer extension product. In the synthesis of a complementary strand to the first primer extension product, e.g. in a PCR reaction, the second strand is extended to the "blocking moiety / stop structure" of the primer structure. Both described primer structures are designed in such a way that the 5 'portion of the primer oligonucleotide remains single-stranded and is not copied by the polymerase.

In a further embodiment, the second region of the primer oligonucleotide comprises a polynucleotide tail having a conventional 5 'to 3' configuration in its entire length and including non-copyable nucleotide modifications. Such non-copyable nucleotide modifications include, for example, 2'-O-alkyl-RNA modifications, PNA, morpholino. These modifications may be distributed differently in the second primer region.

The proportion of non-copyable nucleotide modifications may be between 20% and 100% in the polynucleotide tail, preferably greater than 50% of the nucleotide building blocks. Preferably, these nucleotide modifications are in the 3 'segment of the second region and thus adjacent to the first region of the first primer oligonucleotide.

In one embodiment, the sequence of non-copyable nucleotide modifications is at least partially complementary to the sequence in the template strand such that primer binding to the template occurs involving at least part of these nucleotide modifications. In another embodiment, the sequence of non-copyable nucleotide modifications is non-complementary to the sequence in the template strand.

The non-copyable nucleotide modifications are preferably covalently coupled to one another and thus represent a sequence segment in the second region. The length of this segment comprises between 1 and 40, preferably between 1 and 20 nucleotide modifications, more preferably between 3 and 10 nucleotide modifications.

In a further embodiment, the second region of the first primer oligonucleotide comprises a polynucleotide tail which has a conventional 5'-3 'configuration in its entire length and includes non-copyable nucleotide modifications (eg, 2'-O-alkyl). Modifications) and at least one non-nucleotide linker (eg C3 . C6 , HEG linker). A non-nucleotide linker has the function of covalently linking adjacent nucleotides or nucleotide modifications while at the same time site-specifically disrupting the synthesis function of the polymerase.

Such a non-nucleotide linker should not remove the structures of the polynucleotide tail and the first primer region too far apart. Rather, the polynucleotide tail should be in close proximity to the first primer region. A non-nucleotide linker is taken to include modifications which are no longer than 200 chain atoms in length, more preferably not more than 50 chain atoms, more preferably not more than 10 chain atoms. The minimum length of such a linker can be one atom. An example of such non-nucleotide linkers are straight or branched alkyl linkers having an alkyl chain which includes at least one carbon atom, more preferably at least 2 to 30, more preferably 4 to 18. Such linkers are in oligonucleotide chemistry sufficiently well known (eg C3 . C6 or C12 Linker) and may be introduced during solid-phase synthesis of oligonucleotides between the sequence of the polynucleotide tail and the sequence of the first region of the first primer oligonucleotide. Another example of such non-nucleotide linkers is linear or branched polyethylene glycol derivatives. A well-known example in oligonucleotide chemistry is hexaethylene glycol (HEG). Another example of such non-nucleotide linkers are abasic modifications (eg THF modification, as analog of dRibose).

If one or more of such modifications are integrated into a second region, they can effectively interfere with a polymerase in its copying function during its synthesis of the second primer extension product, so that downstream segments remain uncopied after such modification. The number of such modifications in the second range can be between 1 and 100, preferably between 1 and 10, preferably between 1 and 3.

The position of such a non-nucleotide linker may be at the 3 'end of the second region, thus representing the transition to the first region and the second region of the primer oligonucleotide.

The location of the non-nucleotide linker in the middle segment of the second region may also be used. This divides a polynucleotide tail into at least two segments. In this embodiment, the 3 'segment of the polynucleotide tail includes at least one, more preferably several, e.g. between 2 and 20, preferably between 2 and 10 non-copyable nucleotide modifications. These non-copyable nucleotide modifications are preferably located at the junction between the first and second regions of the primer oligonucleotide.

In a further embodiment, the second region of the primer oligonucleotide comprises a polynucleotide tail having in its entire length an array of 5 'to 3' and at least one nucleotide monomer in a "reverse" array of 3 'to 5 'and which are positioned at the junction between the first and second regions of the first primer oligonucleotide.

In a further embodiment, the second region of the primer oligonucleotide comprises a polynucleotide tail, such polynucleotide tail consisting entirely of nucleotides directly adjacent to the first region of the first primer oligonucleotide in reverse order so that the coupling of the first and the second area through 5'-5 'position. An advantage of such an arrangement is that after copying the first region, the polymerase encounters a "reverse" arrangement of nucleotides, which typically results in termination of the synthesis at that site.

In a "reverse" array of nucleotides in the total length of the polynucleotide tail, preferably the 3'-terminal nucleotide of the polynucleotide tail should be blocked at its 3'-OH end to prevent side reactions. Alternatively, a terminal nucleotide may be used which does not have any 3'-OH group, e.g. a dideoxy nucleotide.

In such an embodiment, of course, the corresponding nucleotide arrangement is to be adapted in the controller oligonucleotide. In such a case, the first and second regions of the controller oligonucleotide must be linked in a 3'-3 'arrangement.

In a further embodiment, the second region of the primer oligonucleotide comprises a polynucleotide tail having in its entire length a conventional arrangement of 5 'to 3' and including at least one nucleotide modification which is not a complementary nucleobase for the polymerase if the synthesis is carried out using exclusively natural dNTP (dATP, dCTP, dGTP, dTTP or dUTP).

For example, iso-dG or iso-dC nucleotide modifications may be integrated as single, but preferably several (at least 2 to 20) nucleotide modifications in the second region of the first primer oligonucleotide. Further examples of nucleobase modifications are various modifications of the extended genetic alphabet. Such nucleotide modifications preferably do not support complementary base pairing with natural nucleotides, such that a polymerase (at least theoretically) does not contain a nucleotide from the series (dATP, dCTP, dGTP, dTTP or dUTP). However, in reality, rudimentary incorporation may occur, especially at higher concentrations of dNTP substrates and prolonged incubation times (e.g., 60 minutes or longer). Therefore, it is preferable to use a plurality of such nucleotide modifications positioned at adjacent sites. The stop of polymerase synthesis is effected by the lack of suitable complementary substrates for these modifications. Oligonucleotides with iso-dC and iso-dG, respectively, can be synthesized by standard techniques and are available from several commercial suppliers (e.g., Trilink-Technologies, Eurogentec, Biomers GmbH). Optionally, the sequence of the first region of the controller oligonucleotide can also be adapted to the sequence of such a second primer region. In this case, complementary nucleobases of the extended genetic alphabet can be integrated into the first region of the controller oligonucleotide accordingly during the chemical synthesis. For example, iso-dG may be integrated in the second region of the first primer nucleotide, its complementary nucleotide (iso-dC-5-Me) may be placed at the appropriate location in the first region of the controller oligonucleotide.

In summary, the termination of the synthesis of polymerase in the second region can be achieved in various ways. However, this blockage preferably takes place only when polymerase has copied the first region of the first primer oligonucleotide. This ensures that a second primer extension product has an appropriate primer binding site in its 3 'segment. This primer binding site is exposed as part of the strand displacement and is thus available for a new binding of another first primer oligonucleotide.

In the synthesis of the complementary strand to the first primer extension product, the primer extension reaction remains in front of the polynucleotide tail. Because this polynucleotide tail remains single stranded for interaction with the controller oligonucleotide and thus is available for binding, it promotes the initiation of the strand displacement reaction by the controller oligonucleotide by placing it in the immediate vicinity of the corresponding complementary segments of the controller oligonucleotide Near the matching duplex-end brings. The distance between the complementary part of the controller oligonucleotide (second region) and the complementary part of the extended primer oligonucleotide (first region) is thereby reduced to a minimum. Such spatial proximity facilitates the initiation of strand displacement.

In the context of a schematic representation of a nucleic acid-mediated strand displacement reaction, a complementary sequence of controller oligonucleotide is now in the immediate vicinity of the appropriate duplex end. There is competition for binding to the first region of the first primer oligonucleotide between the strand of the controller oligonucleotide and the primer complementary template strand. By repetetive closure and formation of base pairing between the primer region and the complementary segment of the controller oligonucleotide (second region of the controller nucleotide) or the complementary segment of the template strand, the initiation of the nucleic acid-mediated strand displacement process occurs.

Generally, the closer the corresponding complementary sequence portion of the controller oligonucleotide to the complementary segment of the primer region is, the higher the yield of initiation of rod displacement. By increasing this distance, however, the yield of the initiation of the strand displacement decreases.

In the context of the present invention, it is not absolutely necessary for the initiation of the strand displacement to work with maximum yield. Therefore, distances between the 5 'segment of the first primer region of the first primer oligonucleotide, which binds to a complementary strand of the template and forms a complementary duplex, and a corresponding one complementary sequence segment in the controller oligonucleotide when bound to polynucleotide tail of the second region of the first primer oligonucleotide in the following ranges: between 0.1 and 20 nm, more preferably between 0.1 and 5 nm, even better between 0.1 and 1 nm. In the preferred case, this distance is less than 1 nm. Expressed in other units, this distance corresponds to a distance of less than 200 atoms, more preferably less than 50 atoms, even better less than 10 atoms. In the preferred case, this distance is an atom. The distance information is for guidance only and illustrates that shorter distances between these structures are preferred. The measurement of this distance is in many cases only possible by analyzing the exact structures of oligonucleotides and measuring the measurement of sequence distances or of linker lengths.

The first primer may also include additional sequence segments that are not required to interact with the controller oligonucleotide or template strand. Such sequence segments may, for example, bind further oligonucleotides which are used as detection probes or immobilization partners when bound to the solid phase.

Primer function of the first primer oligonucleotide

The first primer oligonucleotide can be used in several partial steps. First and foremost, it performs a primer function in the amplification. Here, primer extension reaction is carried out using the second primer extension product as a template. In another embodiment, the first primer oligonucleotide may use the starting nucleic acid chain as a template at the beginning of the amplification reaction. In a further embodiment, the first primer oligonucleotide can be used in the preparation / provision of a starting nucleic acid chain.

In the context of amplification, the first primer serves as the initiator of the synthesis of the first primer extension product using the second primer extension product as a template. The 3 'segment of the first primer comprises a sequence which can bind predominantly complementary to the second primer extension product. Enzymatic extension of the first primer oligonucleotide using the second primer extension product as template results in formation of the first primer extension product. Such a first primer extension product comprises the target sequence or its sequence parts. In the course of the synthesis of the second primer extension product, the sequence of the copiable portion of the first primer oligonucleotide is recognized by polymerase as a template and a corresponding complementary sequence is synthesized to result in a corresponding primer binding site for the first primer oligonucleotide. The synthesis of the first primer extension product occurs until including 5 'segment of the second primer oligonucleotide. Immediately after the synthesis of the first primer extension product, this product is bound to the second primer extension product and forms double-stranded complex. The second primer extension product is sequence-specifically displaced from this complex by the controller oligonucleotide. In doing so, the controller oligonucleotide binds to the first primer extension product. Again, following successful strand displacement by controller oligonucleotide, the second primer extension product may itself serve as a template for the synthesis of the first primer extension product. The now vacant 3 'segment of the first primer extension product can bind another second primer oligonucleotide so that a new synthesis of the second primer extension product can be initiated.

Furthermore, the first primer oligonucleotide may serve as an initiator of the synthesis of the first primer extension product starting from the nucleic acid sequence at the beginning of the amplification. In one embodiment, the sequence of the first primer is completely complementary to the corresponding sequence segment of a starting nucleic acid chain. In a further embodiment, the sequence of the first primer oligonucleotide is only partially complementary to the corresponding sequence segment of a starting nucleic acid chain. However, this divergent complementarity is not intended to prevent the first primer oligonucleotide from starting a predominantly sequence-specific primer extension reaction. The respective differences in complementarity of the first primer oligonucleotide to the respective position in the starting nucleic acid chain are preferably in the 5 'segment of the first region of the first primer oligonucleotide, so that predominantly complementary base pairing and initiation of the synthesis is possible in the 3' segment , For the initiation of the synthesis, for example, especially the first 4-10 positions in the 3 'segment should be fully complementary to the template (starting nucleic acid chain). The remaining nucleotide positions may differ from perfect complementarity. Thus, the extent of perfect complementarity in the remainder of the 5 'segment of the first region of the first primer oligonucleotide can range between 50% to 100%, more preferably between 80% and 100%, of the base composition. Depending on the length of the first region of the first primer oligonucleotide, comprising the sequence deviations from 1 to a maximum of 15 positions, better 1 to a maximum of 5 positions. The first primer oligonucleotide may thus initiate synthesis from a startup nucleic acid chain. In a subsequent synthesis of the second primer extension product, copyable sequence segments of the first primer oligonucleotide are copied from polymerase, so that again in subsequent synthesis cycles, a fully complementary primer binding site is formed within the second primer extension product for binding of the first primer oligonucleotide and is available in subsequent synthesis cycles.

In another embodiment, the first primer oligonucleotide may be used in the preparation of a starting nucleic acid chain. In so doing, such a first primer oligonucleotide may bind predominantly / preferably sequence specifically to a nucleic acid (e.g., a single-stranded genomic DNA or RNA or its equivalents comprising a target sequence) and initiate a template-dependent primer extension reaction in the presence of a polymerase. The binding position is chosen such that the primer extension product comprises a desired target sequence. The extension of the first primer oligonucleotide results in a nucleic acid strand which has a template-complementary sequence. Such a strand may be detached from the template (e.g., by heat or alkali) and thus converted to a single-stranded form. Such a single-stranded nucleic acid chain can serve as a starting nucleic acid chain at the beginning of the amplification. Such a start nucleic acid chain comprises in its 5 'segment the sequence portions of the first primer oligonucleotide, furthermore it comprises a target sequence or its equivalents and a primer binding site for the second primer oligonucleotide. Further steps are explained in the section "Starting nucleic acid chain".

The synthesis of the first primer extension product is a primer extension reaction and forms a partial step in the amplification. The reaction conditions during this step are adjusted accordingly. The reaction temperature and the reaction time are chosen so that the reaction can take place successfully. The particular preferred temperature in this step depends on the polymerase used and the binding strength of the respective first primer oligonucleotide to its primer binding site and includes, for example, ranges from 15 ° C to 75 ° C, more preferably from 20 to 65 ° C, preferably from 25 ° C to 65 ° C. The concentration of the first primer oligonucleotide comprises ranges from 0.01 μmol / L to 50 μmol / L, more preferably from 0.1 μmol / L to 20 μmol / L, preferably from 0.1 μmol / L to 10 μmol / L.

In one embodiment, all steps of the amplification run under stringent conditions which prevent or slow down formation of nonspecific products / by-products. Such conditions include, for example, higher temperatures, for example above 50 ° C.

If more than one specific nucleic acid chain has to be amplified in one batch, in one embodiment preferably sequence-specific primer oligonucleotides are used for amplification of corresponding respective target sequences.

Preferably, sequences of the first, second primer oligonucleotide and the controller oligonucleotide are matched to one another such that side reactions, e.g. Primer-dimer formation, minimized. For this purpose, for example, the sequence of the first and second primer oligonucleotides are adapted to each other such that both primer oligonucleotides are incapable of undergoing an amplification reaction in the absence of an appropriate template and / or target sequence and / or starting sequence. Start or support nucleic acid chain. This can be achieved, for example, in that the second primer oligonucleotide does not comprise a primer binding site for the first primer oligonucleotide and the first primer oligonucleotide does not comprise a primer binding site for the second primer oligonucleotide. Furthermore, it should be avoided that the primer sequences comprise extended self-complementary structures (self-complement).

In one embodiment, the synthesis of the first and second primer extension products proceeds at the same temperature. In another embodiment, the synthesis of the first and second primer extension products proceeds at different temperatures. In another embodiment, the synthesis of the first primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature. In another embodiment, synthesis of the first primer extension product and strand displacement by controller oligonucleotide proceeds at different temperatures.

Preferred embodiments of the controller oligonucleotide

• • Einen ersten einzelsträngigen Bereich, welcher an den Polynukleotid-Schwanz des zweiten Bereichs des ersten Primer-Oligonukleotides binden kann,
• • einen zweiten einzelsträngigen Bereich, welcher an den ersten Bereich des ersten Primer-Oligonukleotides im wesentlich komplementär binden kann,
• • einen dritten einzelsträngigen Bereich, welcher zumindest zu einem Segment des Verlängerungsprodukts des ersten Primer-Verlängerungsprodukts im Wesentlichen komplementär ist,

und das Controller-Oligonukleotid dient nicht als Matrize für Primerverlängerung des ersten oder des zweiten Primer-Oligonukleotids.A controller oligonucleotide ( 19 ) comprises:

• A first single-stranded region which can bind to the polynucleotide tail of the second region of the first primer oligonucleotide,
• A second single-stranded region which can bind substantially complementary to the first region of the first primer oligonucleotide,
• A third single-stranded region which is substantially complementary to at least one segment of the extension product of the first primer extension product,

and the controller oligonucleotide does not serve as a template for primer extension of the first or second primer oligonucleotide.

In general, the sequence of the third region of the controller oligonucleotide is adapted to the sequence of the nucleic acid to be amplified, since this is a template for the order of the nucleotides in the extension product of a first primer. The sequence of the second region of controller oligonucleotide is adapted to the sequence of the first primer region. The structure of the first region of the controller oligonucleotide is adapted to the sequence of the second region of the first primer oligonucleotide, especially the nature of the polynucleotide tail.

A controller oligonucleotide may also include other sequence segments that do not belong to the first, second or third region. These sequences can be attached, for example, as flanking sequences at the 3 'and 5' ends. Preferably, these sequence segments do not interfere with the function of the controller oligonucleotide.

The structure of the controller oligonucleotide preferably has the following properties:

The individual areas are covalently bonded to each other. The binding can be done for example via conventional 5'-3 'binding. For example, a phospho-diester bond or a nuclease-resistant phospho-thioester bond can be used.

A controller oligonucleotide may bind by its first region to the polynucleotide tail of the first primer oligonucleotide, which binding is mediated primarily by hybridization of complementary bases. The length of this first region is 3 to 80 nucleotides, preferably 4 to 40 nucleotides, more preferably 6 to 20 nucleotides. The degree of sequence match between the sequence of the first region of the controller oligonucleotide and the sequence of the second region of the first primer oligonucleotide may be between 20% and 100%, preferably between 50% and 100%, most preferably between 80% and 100% , The binding of the first region of the controller oligonucleotide should preferably be made specifically to the second region of the first primer oligonucleotide under reaction conditions.

The sequence of the first region of the controller oligonucleotide is preferably selected such that the number of complementary bases capable of complementary binding to the second region of the first primer oligonucleotide is between 1 and 40, more preferably between 3 and 20, preferably between 6 and 15 lies.

Since the controller oligonucleotide is not a template for polymerase, it may include nucleotide modifications that do not support polymerase function, which may be both base modifications and / or sugar-phosphate backbone modifications. The controller oligonucleotide may include, for example, in its first region, nucleotides and / or nucleotide modifications selected from the following list: DNA, RNA, LNA ("locked nucleic acids" analogs having 2'-4 'bridge linkage in the sugar moiety), UNA ("unlocked nucleid acids" without binding between 2'-3'-atoms of the sugar residue), peptide nucleic acid analogs (PNA), phosphorothioate (PTO), morpholino analogues, 2'-O-alkyl-RNA modifications ( such as 2'-OMe, 2'-O propargyl, 2'-O- (2-methoxyethyl), 2'-O-propyl-amine), 2'-halogen-RNA, 2'-amino-RNA, etc. These nucleotides or nucleotide modifications are linked together, for example, by conventional 5'-3 'or 5'-2' binding. For example, a phospho-diester bond or a nuclease-resistant phospho-thioester bond can be used.

The controller oligonucleotide may include nucleotides and / or nucleotide modifications in its first region, the nucleobases being selected from the following list: adenines and their analogs, guanines and its analogs, cytosines and its analogs, uracil and its analogs, thymines and his Analogues, inosines or other universal bases (eg nitroindole), 2-amino-adenine and its analogues, iso-cytosines and its analogues, iso-guanines and its analogues.

The controller oligonucleotide may include in its first region non-nucleotide compounds selected from the following list: Intercalators which may affect binding strength between the controller oligonucleotide and the first primer oligonucleotide, e.g. MGB, naphthalene etc. The same elements can also be used in the second region of the first primer.

The controller oligonucleotide may include in its first region non-nucleotide compounds, eg, linkers such as C3 . C6 , HEG Linker, which link individual segments of the first area together.

The controller oligonucleotide may bind by means of its second region to the first primer region of the first primer oligonucleotide, the binding being mediated essentially by hybridization of complementary bases.

The length of the second region of the controller oligonucleotide is matched to the length of the first region of the first primer oligonucleotide and is preferably consistent therewith. It is between about 3-30 nucleotides, preferably between 5 and 20 nucleotides. The sequence of the second region of controller oligonucleotide is preferably complementary to the first region of the first primer oligonucleotide. The degree of match in complementarity is between 80% and 100%, preferably between 95% and 100%, preferably 100%. The second region of controller oligonucleotide preferably includes nucleotide modifications which prevent polymerase upon extension of the first primer oligonucleotide but do not block or substantially prevent formation of complementary double strands, for example 2'-O-alkyl RNA analogs (eg 2 '). -O-Me, 2'-O- (2-methoxyethyl), 2'-O-propyl, 2'-O-propargyl nucleotide modifications), LNA, PNA or morpholino nucleotide modifications. Single nucleotide monomers are preferably linked via 5'-3 'linkage, but alternative 5'-2' binding between nucleotide monomers may also be used.

The sequence length and nature of the first and second regions of the controller oligonucleotide are preferably selected to be such that binding of these regions to the first primer oligonucleotide is reversible under reaction conditions, at least in one reaction step of the process. This means that although the controller oligonucleotide and the first primer oligonucleotide can bind specifically to one another, this bond should not lead to the formation of a permanently stable reaction complex under conditions of both elements.

Rather, an equilibrium between a bound complex form of controller oligonucleotide and the first primer oligonucleotide and a free form of individual components under reaction conditions should be sought or made possible at least in one reaction step. This ensures that at least a portion of the first primer oligonucleotides are in free form under reaction conditions and can interact with the template to initiate a primer extension reaction. On the other hand, it will be ensured that corresponding sequence regions of the controller oligonucleotide are available for binding with an extended primer oligonucleotide.

By choosing the temperature during the reaction, the proportion of free, single-stranded and thus reactive components can be influenced: by lowering the temperature, first primer oligonucleotides bind to the controller oligonucleotides, so that both participants bind a complementary double-stranded complex. This can be used to lower the concentration of single-stranded forms of individual components. An increase in temperature can lead to dissociation of both components into single-stranded form. In the region of the melting temperature of the complex (controller oligonucleotide / first primer oligonucleotide) are about 50% of the components in single-stranded form and about 50% as a double-stranded complex. By using appropriate temperatures, the concentration of single-stranded forms in the reaction mixture can thus be influenced.

In embodiments of the amplification process comprising temperature changes between individual reaction steps, desired reaction conditions can be brought about during corresponding reaction steps. For example, by using temperature ranges approximately at the level of melting temperature of complexes of controller oligonucleotide / first primer oligonucleotide shares of each free forms of individual components can be influenced. In this case, temperature used leads to destabilization of complexes comprising controller oligonucleotide / first primer Oligonucleotide such that during this reaction step, individual complex components become, at least temporarily, single-stranded and thus able to interact with other reactants. For example, the first sequence region of the controller oligonucleotide may be released from the double-stranded complex with a non-extended first primer and thus may interact with the second sequence region of an extended first primer oligonucleotide, thereby initiating strand displacement. On the other hand, release of a first, non-extended primer oligonucleotide from a complex comprising controller oligonucleotide / first primer oligonucleotide results in the first primer region becoming single-stranded and thus capable of interacting with the template such that primer extension by a primer oligonucleotide Polymerase can be initiated.

The temperature used does not have to correspond exactly to the melting temperature of the complex of controller oligonucleotide / first primer oligonucleotide. It is sufficient if temperature is used in a reaction step, for example in the range of the melting temperature. For example, the temperature in one of the reaction steps comprises ranges of Tm +/- 10 ° C, better Tm +/- 5 ° C, preferably Tm +/- 3 ° C of the complex of controller oligonucleotide / first primer oligonucleotide.

Such a temperature can be set, for example, in the context of the reaction step, which comprises a sequence-specific strand displacement by the controller oligonucleotide.

In embodiments of the amplification process which do not involve temperature cycling between individual reaction steps and amplification under isothermal conditions, reaction conditions are maintained over the entire duration of the amplification reaction in which there is equilibrium between a complex form of controller oligonucleotide and the first primer. Oligonucleotide and a free form of individual components is possible.

The relationship between a complex form of controller oligonucleotide and the first primer oligonucleotide and free forms of individual components can be affected by both reaction conditions (e.g., temperature and Mg 2+ concentration) and by structures and concentrations of individual components.

The sequence length and the nature of the first and second regions of the controller oligonucleotide are, in one embodiment, chosen such that, under given reaction conditions (eg, in the step of strand displacement by the controller oligonucleotide), the ratio between a proportion of free controller oligonucleotide and a portion of a controller oligonucleotide complexed with a first primer oligonucleotide comprises the following ranges: from 1: 100 to 100: 1, preferably from 1:30 to 30: 1, more preferably from 1:10 to 10: 1. The ratio between a proportion of a free first primer oligonucleotide and a proportion of a first primer oligonucleotide complexed with a controller oligonucleotide ranges from 1: 100 to 100: 1, preferably from 1:30 to 30: 1, more preferably from 1:10 to 10: 1.

In one embodiment, the concentration of the first primer oligonucleotide is higher than the concentration of the controller oligonucleotide. As a result, there is an excess of the first primer in the reaction and controller oligonucleotide must be released for its effect of binding with the first primer by choosing the appropriate reaction temperature. In general, this is done by raising the temperature to sufficient levels of free forms of the controller oligonucleotide.

In another embodiment, the concentration of the first primer oligonucleotide is lower than the concentration of the controller oligonucleotide. As a result, there is an excess of the controller oligonucleotide and the first primer oligonucleotide must be released for its effect from binding to the controller oligonucleotide by choosing the appropriate reaction temperature. In general, this is done by raising the temperature to sufficient levels of free forms of the first primer oligonucleotide.

In isothermal conditions, there is an equilibrium: certain portions of the first primer oligonucleotide and controller oligonucleotide are bound together, while others are present as a single-stranded form in the reaction.

The controller oligonucleotide may bind by means of its third region to at least one segment of the specifically synthesized extension product of the first primer oligonucleotide ( 20 to 30 ). Binding is preferably by hybridization of complementary bases between the controller oligonucleotide and the polymerase synthesized extension product.

In order to support the strand displacement reaction, the sequence of the third region should preferably have a high complementarity to the extension product. In one embodiment, the sequence of the third region is 100% complementary to the extension product.

The binding of the third region is preferably carried out on the segment of the extension product which directly adjoins the first region of the first primer oligonucleotide. Thus, the segment of the extension product is preferably in the 5 'segment of the entire extension product of the first primer oligonucleotide.

The binding of the third region of the controller oligonucleotide preferably does not occur over the entire length of the extension product of the first primer oligonucleotide. Preferably, a segment remains unbound at the 3 'end of the extension product. This 3 'segment is necessary for the binding of the second primer oligonucleotide.

The length of the third region is adapted in such a way that the third region binds to the 5 'continuous segment of the extension product on the one hand, and on the other hand does not bind the 3' continuous segment of the extension product.

The total length of the third region of the controller oligonucleotide is from 2 to 100, preferably from 6 to 60, more preferably from 10 to 40 nucleotides or their equivalents. The controller oligonucleotide may bind to the segment of the extension product along that length, thereby displacing this 5 'segment of the extension product from binding with its complementary template strand.

The length of the 3'-segment of the extension product that is not bound by the controller oligonucleotide includes, for example, ranges between 5 and 100 nucleotides, more preferably 5 and 60 nucleotides, preferably between 10 and 40, preferably between 15 and 30 nucleotides.

This 3 'continuous segment of the extension product is not displaced from the controller oligonucleotide from binding with the template strand. Even with the third region of the controller oligonucleotide fully bound to its complementary segment of the extension product, the first primer extension product may remain bound to the template strand via its 3'-term segment. The bond strength of this complex is preferably selected so that it can dissociate spontaneously under reaction conditions (step e)), for example. This can be achieved, for example, by the melting temperature of this complex from the 3'-segment of the extension product of the first primer oligonucleotide and its template strand being approximately in the range of the reaction temperature or below the reaction temperature in a corresponding reaction step (reaction step e). With little stability of this complex in the 3 'segment of the extension product, complete binding of the third region of the controller oligonucleotide to the 5' segment of the extension product results in rapid dissociation of the first primer extension product from its template strand.

The controller oligonucleotide as a whole has a suitable structure to perform its function: under appropriate reaction conditions, it is capable of sequence-specific displacement of the extended first primer oligonucleotide from binding with the template strand, thereby converting the template strand into single-stranded form, and thus for further binding with a novel first primer oligonucleotide and their target sequence-specific extension by polymerase.

To fulfill the strand displacement function, regions one, two and three of the controller oligonucleotide should be predominantly in single-stranded form under reaction conditions. Therefore, double-stranded self-complementary structures (e.g., hairpins) in these regions should be avoided as much as possible because they may degrade functionality of the controller oligonucleotide.

The controller oligonucleotide should not occur as a template in the method of the present invention, therefore, the first primer oligonucleotide, when attached to the controller oligonucleotide under reaction conditions, should not be extended by polymerase.

This is preferably achieved by using nucleotide modifications which prevent the polymerase from copying the strand. Preferably, the 3 'end of the first primer oligonucleotide does not remain elongated when the first primer oligonucleotide binds to the controller oligonucleotide under reaction conditions.

The degree of blockage / inhibition / slowing / aggravation of the reaction may be between complete expression of this property (e.g., 100% blockage under given reaction conditions) and a partial expression of this property (e.g., 30-90% blockade under given reaction conditions). Preferred are nucleotide modifications which individually or in a series coupled to each other (eg as a sequence fragment consisting of modified nucleotides) more than 70%, preferably more than 90%, more preferably more than 95%, and particularly preferably 100% extension of a first primer can prevent.

The nucleotide modifications may include base modifications and / or sugar-phosphate-residue modifications. The sugar-phosphate modifications are preferred because any complementary sequence of a controller oligonucleotide can be assembled by combining with conventional nucleobases. The nucleotides with modifications in the sugar-phosphate residue, which can lead to obstruction or blockade of the synthesis of the polymerase include, for example: 2'-O-alkyl modifications (eg 2'-O-methyl, 2'-O- (2-methoxyethyl), 2'-O-propyl, 2'-O-propargyl nucleotide modifications), 2'-amino-2'-deoxy-nucleotide modifications, 2'-amino-alkyl-2'-deoxy- Nucleotide modifications, PNA, morpholino modifications, etc.

The blockade can be accomplished by either a single nucleotide modification or only by coupling multiple nucleotide modifications in series (e.g., as a sequence fragment consisting of modified nucleotides). For example, at least 2, preferably at least 5, more preferably at least 10 such nucleotide modifications may be coupled side by side in the controller oligonucleotide.

A controller oligonucleotide may comprise a uniform type of nucleotide modifications or may comprise at least two different types of nucleotide modification.

The location of such nucleotide modifications in the controller oligonucleotide is preferably intended to prevent the polymerase from extending the 3 'end of a first primer oligonucleotide bound to the controller oligonucleotide.

In one embodiment, such nucleotide modifications are located in the second region of the controller oligonucleotide. In another embodiment, such nucleotide modifications are located in the third region of the controller oligonucleotide. In another embodiment, such nucleotide modifications are located in the second and third regions of the controller oligonucleotide.

For example, the second region of the controller oligonucleotide consists of at least 20% of its positions from such nucleotide modifications, preferably at least 50%.

For example, the third region of the controller oligonucleotide consists of at least 20% of its positions from such nucleotide modifications, preferably at least 50%, more preferably at least 90%. In one embodiment, the entire third region comprises nucleotide modifications that prevent a polymerase from extending a primer bound to such regions using the controller oligonucleotide as a template. In a further embodiment, the entire third and second region comprises such nucleotide modifications. In a further embodiment, the entire first, second and third region comprises such nucleotide modifications. Thus, the controller oligonucleotide may consist entirely of such nucleotide modifications. Such modified controller oligonucleotides can be used, for example, in multiplex analyzes in which further primers are used. This is to prevent inadvertent primer extension reactions on one or more controller oligonucleotides.

The sequence of nucleobases from these nucleotide modifications is adapted to the requirements of the sequence in each area.

For example, the remaining portion may be natural nucleotides, or nucleotide modifications that do not or only marginally inhibit polymerase function, eg, DNA nucleotides. PTO nucleotides, LNA nucleotides, RNA nucleotides. In this case, further modifications, for example base modifications such as 2-amino-adenosine, 2-aminopurines, 5-methyl-cytosines, inosines, 5-nitroindoles, 7-deaza-adenosine, 7-deaza-guanosine, 5-propyl-cytosine, 5 Propyl uridine or non-nucleotide modifications such as dyes, or MGB modifications, etc. can be used, for example, to match binding strengths of individual regions of the controller oligonucleotides. The individual nucleotide monomers can be coupled to one another via conventional 5'-3 'binding or also deviating via 5'-2' binding.

A segment of the controller oligonucleotide nucleotide with nucleotide modifications that prevent polymerase extension of the 3 'end of a primer oligonucleotide bound to controller oligonucleotide is referred to as a "second blocking moiety." The length of this segment may include from 1 to 50 nuclide modifications, preferably between 4 and 30. For example, this segment may be located in the controller oligonucleotide such that the 3 'end of the bound first primer oligonucleotide is in this segment. Thus, this segment can span regions two and three.

In one embodiment, preferably no linker structures or spacer structures, such as C3 . C6 , HEG linker used to prevent extension of the 3 'end of a first primer oligonucleotide bound to the controller oligonucleotide.

In one embodiment, a controller oligonucleotide in its third region comprises at least one component of the detection system (eg fluorescence reporter or fluorescence quencher or a donor fluorophore.) In one embodiment, the position of this component is at the 5 'end of the controller oligonucleotide In another embodiment, this component is located in the inner segment of the third region, wherein the distance to the 5 'end of the controller oligonucleotide may be between 2 to 50 nucleotides, more preferably between 2 and 20, preferably between 2 and 10 nucleotides the oligonucleotide probe and the controller oligonucleotide on the same first primer extension product, it comes to a spatial proximity of the two components of the detection system (eg between a fluorescent reporter on the oligonucleotide probe and a donor fluorophore on the controller oligonucleotide).

The controller oligonucleotide may comprise, in addition to regions one, two and three, further sequence segments which, for example, flank the abovementioned regions in the 5 'segment or 3' segment of the controller oligonucleotide. Such sequence elements may be used, for example, for other functions, such as interaction with probes, binding to solid phase, etc. Such regions preferably do not interfere with the function of regions one to three. The length of these flanking sequences may be, for example, between 1 to 50 nucleotides. Furthermore, a controller oligonucleotide may comprise at least one element for immobilization on a solid phase, e.g. a biotin residue. Furthermore, a controller oligonucleotide may comprise at least one element for detection, e.g. a fluorescent dye.

• • Bei korrekter Übereinstimmung mit vorgegebenen Sequenzinhalten des Controller-Oligonukleotides erfolgt eine Strangverdrängung, wobei die Matrizen-Stränge von neusynthetisierten Strängen abgelöst werden. Dabei werden Primer-Bindungsstellen in einzelsträngigen Zustand überführt und stehen für neue Interaktion mit Primern und somit für eine weitere Synthese zur Verfügung. Somit wird das System aus beiden Primer-Verlängerungsprodukten in einen aktiven Zustand versetzt. Das Controller-Oligonukleotid hat somit eine aktivierende Wirkung auf das System.
• • Bei fehlender Übereinstimmung mit vorgegebenen Sequenzinhalten des Controller-Oligonukleotides wird Strangverdrängung der Matrizen-Stränge von neusynthetisierten Strängen beieinflusst. Die Strangverdrängung und /oder Ablösung wird entweder quantitativ verlangsamt oder komplett aufgehoben. Es kommt somit gar nicht oder seltener zu Überführung von Primer-Bindungsstellen in einzelsträngigen Zustand. Somit stehen für neue Interaktion mit Primern gar keine oder quantitativ gesehen wenige Primer-Bindungsstellen zur Verfügung. Somit wird das System aus beiden Primer-Verlängerungsprodukten seltener in einen aktiven Zustand versetzt bzw. aktiver Zustand wird gar nicht erreicht.

In the presence of a controller oligonucleotide, newly synthesized sequences are checked for their sequence contents by interaction with the controller oligonucleotide.

• • If correctly matched with given sequence contents of the controller oligonucleotide, strand displacement occurs, with the template strands being replaced by newly synthesized strands. Here, primer binding sites are transformed into single-stranded state and are available for new interaction with primers and thus for further synthesis. Thus, the system of both primer extension products is placed in an active state. The controller oligonucleotide thus has an activating effect on the system.
• In the absence of conformance to given sequence contents of the controller oligonucleotide, strand displacement of the template strands of newly synthesized strands is affected. The strand displacement and / or separation is either slowed down quantitatively or completely eliminated. There is thus no or less often to transfer of primer binding sites in single-stranded state. Thus, there are no or only a few primer binding sites available for new interaction with primers. Thus, the system of both primer extension products is less frequently put into an active state or active state is not reached at all.

The efficiency of the double-stranded opening of the newly synthesized primer extension products after each individual synthesis step affects the potentially achievable yields in subsequent cycles: the fewer free / single-stranded primer binding sites of a nucleic acid chain to be amplified at the beginning of a synthesis step The lower the number of newly synthesized strands of the nucleic acid chain to be amplified in this step. In other words: the Yield of a synthesis cycle is proportional to the amount of primer binding sites available for interaction with corresponding complementary primers. Overall, this can be realized a control circuit.

This control circuit corresponds to a real-time / on-line control of synthesized fragments: Sequence control takes place in the reaction mixture while amplification takes place. This sequence control follows a predetermined pattern and the oligonucleotide system (by strand-opening action of controller oligonucleotide) can decide between "correct" and "non-correct" states without external interference. In the correct state, the synthesis of sequences is continued; in the incorrect state, the synthesis is either slowed down or completely prevented. The resulting differences in yields of "correct" and "non-correct" sequences after each step affect the entire amplification comprising a variety of such steps.

In exponential amplification, this dependence is exponential so that even slight differences in efficiency in a single synthesis cycle due to sequence differences may signify a significant time delay in total amplification, or cause the complete absence of detectable amplification within a given time frame.

This effect of the real-time control of the newly synthesized nucleic acid chains is associated with the use of the controller oligonucleotide and the influence of controller oligonucleotide in the context of an amplification thus goes significantly beyond the length of primer oligonucleotides.

Reaction conditions in strand displacement reaction

The displacement of the second primer extension product from binding with the first primer extension product by means of a sequence-dependent strand displacement by the controller oligonucleotide forms a partial step in the amplification. The reaction conditions during this step are adjusted accordingly. The reaction temperature and the reaction time are chosen so that the reaction can take place successfully.

In a preferred embodiment, the strand displacement by the controller oligonucleotide until detachment / dissociation of the second primer extension product from binding with the first primer extension product. Such dissociation of the 3 'segment of the first primer extension product from complementary portions of the second primer extension product may be spontaneous as part of a temperature dependent / temperature related separation of both primer extension products. Such dissociation has a favorable effect on the kinetics of the amplification reaction and may be influenced by the choice of reaction conditions, e.g. by means of temperature conditions. The temperature conditions are therefore chosen such that successful strand displacement by complementary binding of the controller oligonucleotide favors dissociation of the second primer extension product from the 3 'segment of the first primer extension product.

In a further preferred embodiment, the strand displacement through the controller oligonucleotide proceeds until detachment / dissociation of a 3 'segment of the second primer extension product ( P2.1 -Ext) from the complementary binding with the first primer extension product ( P1.1 ), This 3 'segment of the second primer extension product ( P2.1 ) Comprises at least one complementary region to the first primer and a complementary segment to the first primer extension product ( P1.1 -Ext), which was formed only in enzymatic synthesis. This leads to the formation of a complex (C1.1 / P1.1 -Ext / P2.1 .-Ext) ( 1B) containing both the first primer extension product ( P1.1 -Ext), the second primer extension product ( P2.1 -Ext) and the controller oligonucleotide ( C1.1 ). In such a complex, the 3 'segment of the second primer extension product is at least transiently single-stranded since it can be displaced from the controller oligonucleotide from its binding with the first primer extension product. Schematically represented, there is a balance between binding and detachment of the P2.1 -Ext to the P1.1 -Ext. The 3 ' Segment of the first primer extension product ( P1.1 ) Is in such a complex complementary hybridized to the second primer extension product ( P2.1 -Ext) ( 1B) ,

Due to a partially open primer binding site for the first primer oligonucleotide (3 'segment of the second primer extension product), a new primer extension product ( P1.2 ) to a single-stranded sequence segment of the complex ( P2.1 -Ext) under reaction conditions and thus a synthesis of a new first primer extension product ( P1.2 -ext) initiated by a polymerase. In general, this reaction proceeds at a reduced rate, since 3 'segment of the P2.1 -Ext is not permanently single-stranded, but is in the competitive behavior with controller oligonucleotide and thus alternately single-stranded and double-stranded states by binding to the P1.1 -Ext has.

Continuation of this newly started synthesis of P1.2 -Ext using the second primer extension product as a template ( P2.1 -Ext) may also cause dissociation of the complex by polymerase-induced strand displacement ( C1.1 / P1.1 -Ext / P2.1 Ext.). Controller oligonucleotide, temperature-dependent double-strand destabilization and strand displacement by the polymerase act synergistically and complementarily. This results in a dissociation of the 3 'segment of the first primer extension product ( P1.1 -Ext) of complementary portions of the second primer extension product ( P2.1 -Ext).

Such dissociation has a favorable effect on the kinetics of the amplification reaction and can be influenced by the choice of reaction conditions, eg by means of temperature conditions. The involvement of polymerase-mediated synthesis-dependent strand displacement in the dissociation of P1.1 -Ext and P2.1 -Ext has a favorable effect on strand separation.

The temperature in this step includes, for example, ranges from 15 ° C to 75 ° C, more preferably from 30 ° C to 70 ° C, preferably from 50 ° C to 70 ° C.

For a given length of the first region of the controller oligonucleotide and the second region of the first primer oligonucleotide (comprising, for example, ranges of from 3 to 25 nucleotide monomers, more preferably from 5 to 15 nucleotide monomers), a strand displacement reaction can generally be successfully initiated. With complete complementarity of the controller oligonucleotide to corresponding portions of the first primer extension product, the controller oligonucleotide can bind to the first primer extension product to the 3 'segment of the first primer extension product and displace the second primer extension product. The second primer extension product thus remains in association with the 3 'segment of the first primer extension product. This strength of this compound can be influenced by temperature. Upon reaching a critical temperature, this compound can decay and dissociate both primer extension products. The shorter the sequence of the 3 'segment, the more unstable this compound and the lower the temperature which causes spontaneous dissociation.

A spontaneous Dissozialtion can be achieved for example in the temperature range, which is approximately at the melting temperature. In one embodiment, the temperature of the strand displacement steps through the controller oligonucleotide is at about the melting temperature (Tm +/- 3 ° C) of the complex comprising the 3 'segment of the first primer extension product that is not bound by controller oligonucleotide , and the second primer oligonucleotide and the second primer extension product, respectively.

In one embodiment, the temperature of the strand displacement steps through the controller oligonucleotide is at about the melting temperature (Tm +/- 5 ° C) of the complex comprising the 3 'segment of the first primer extension product that is not bound by controller oligonucleotide , and the second primer oligonucleotide and the second primer extension product, respectively.

In one embodiment, the temperature of the steps of strand displacement by the controller oligonucleotide is above the melting temperature of the complex comprising the 3 'segment of the first primer extension product not bound by the controller oligonucleotide and the second primer oligonucleotide second primer extension product. Such a temperature includes temperature ranges from about Tm + 5 ° C to Tm + 20 ° C, better from Tm + 5 ° C to Tm + 10 ° C. By using a higher temperature, the equilibrium in this reaction step can be shifted towards dissociation. As a result, the kinetics of the reaction can be favorably influenced. Use of too low temperatures in the strand displacement step by means of controller oligonucleotide can lead to a significant slowing of the amplification.

In one embodiment, a first primer extension product comprises a 3 'segment which is not bound by controller oligonucleotide and which comprises sequence lengths of 9 to about 18 nucleotides. In this embodiment, spontaneous dissociation can usually be achieved already at temperature ranges between 40 ° C and 65 ° C. Higher temperatures also lead to dissociation.

In one embodiment, a first primer extension product comprises a 3 'segment which is not bound by controller oligonucleotide and which comprises sequence lengths of 15 to about 25 nucleotides. In this embodiment, spontaneous dissociation can usually be achieved already at temperature ranges between 50 ° C and 70 ° C. Higher temperatures also lead to dissociation.

In one embodiment, a first primer extension product comprises a 3 'segment which is not bound by controller oligonucleotide and which comprises sequence lengths of from 20 to about 40 nucleotides. In this embodiment, a spontaneous dissociation usually already at temperature ranges between 50 ° C and 75 ° C can be achieved. Higher temperatures also lead to dissociation.

The composition of the 3 'segment of the first primer extension product and optionally introduction of melting temperature affecting oligonucleotide modifications (e.g., MGB) or reaction conditions (e.g., TPAC, betaines) may affect the choice of temperature. Appropriate adaptation can therefore be made.

In one embodiment, all steps of the amplification run under stringent conditions which prevent or slow down formation of nonspecific products / by-products. Such conditions include, for example, higher temperatures, for example above 50 ° C.

In one embodiment, the single steps of strand displacement by controller oligonucleotides proceed at the same temperature as the synthesis of the first and second primer extension products. In a further embodiment, the individual steps of strand displacement by controller oligonucleotides proceed at temperature which deviates from the temperature of the respective synthesis of the first and the second primer extension product. In another embodiment, the synthesis of the first primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature. In another embodiment, synthesis of the second primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature.

The concentration of the controller oligonucleotide ranges from 0.01 μmol / L to 50 μmol / L, more preferably from 0.1 μmol / L to 20 μmol / L, preferably from 0.1 μmol / L to 10 μmol / L.

Preferred Embodiments of the Second Primer Oligonucleotide (Primer-2):

An oligonucleotide capable of binding with its 3 'segment to a substantially complementary sequence within the nucleic acid or its equivalents to be amplified and initiating a specific second primer extension reaction ( 22 to 25 ). This second primer oligonucleotide is thus capable of binding to the 3 'segment of a first specific primer extension product of the first primer oligonucleotide and initiating a polymerase-dependent synthesis of a second primer extension product. In one embodiment, every second primer oligonucleotide is specific for each nucleic acid to be amplified.

The second primer oligonucleotide should be able to be copied in reverse synthesis and also serves as a template for the synthesis of the first primer extension product.

The length of the second primer oligonucleotide may be between 15 and 100 nucleotides, preferably between 20 and 60 nucleotides, more preferably between 30 and 50 nucleotides. The nucleotide building blocks are preferably linked to one another via customary 5'-3 'phosphodiester bond or phosphothioester bond. Such a primer oligonucleotide can be chemically synthesized in the desired form.

• • natürliche Nukleotide (dA, dT, dC, dG etc.) oder deren Modifikationen ohne veränderte Basen-Paarung
• • Modifizierte Nukleotide, 2-Amino-dA, 2-thio-dT oder andere Nukleotid-Modifikationen mit abweichender Basen-Paarung (z.B. Universal-Basenpaaren, wie beispielsweise Inosine 5-Nitroindol).

In one embodiment, the second primer oligonucleotide may include nucleotide monomers that do not or only insignificantly affect the function of the polymerase, including, for example:

• • natural nucleotides (dA, dT, dC, dG etc.) or their modifications without altered base pairing
• Modified nucleotides, 2-amino-dA, 2-thio-dT or other nucleotide modifications with different base pairing (eg universal base pairs such as inosine 5-nitroindole).

In a preferred embodiment, the 3'-OH end of this range is preferably free of modification and has a functional 3'-OH group which can be recognized by polymerase and extended template-dependent. In a further preferred embodiment, the 3 'segment of the second comprises Primers at least one phosphorothioate compound, so that no degradation of 3'-end of the primers can be done by 3'-exonuclease activity of polymerases.

The second primer oligonucleotide can be used in several partial steps. First and foremost, it performs a primer function in the amplification. Here, primer extension reaction is carried out using the first primer extension product as a template. In another embodiment, the second primer oligonucleotide may use the starting nucleic acid sequence as a template at the beginning of the amplification reaction. In a further embodiment, the second primer oligonucleotide may be used in the preparation / provision of a starting nucleic acid chain.

In the context of amplification, the second primer serves as the initiator of the synthesis of the second primer extension product using the first primer extension product as template. The 3 'segment of the second primer comprises a sequence which can bind predominantly complementary to the first primer extension product. Enzymatic extension of the second primer oligonucleotide using the first primer extension product as template results in formation of the second primer extension product. Such synthesis typically occurs in parallel with displacement of the controller oligonucleotide from its binding with the first primer extension product. This displacement occurs predominantly by polymerase and can be done partially by the second primer oligonucleotide. Such a second extension product comprises the target sequence or its segments. In the course of the synthesis of the second primer extension product, the sequence of the copiable portion of the first primer oligonucleotide is recognized by polymerase as a template and a corresponding complementary sequence is synthesized. This sequence resides in the 3 'segment of the second primer extension product and comprises primer binding site for the first primer oligonucleotide. The synthesis of the second primer extension product occurs until the stop position in the first primer oligonucleotide. Immediately after the synthesis of the second primer extension product, this product is bound to the first primer extension product and forms a double-stranded complex. The second primer extension product is sequence-specifically displaced from this complex by the controller oligonucleotide. Again, following successful strand displacement by controller oligonucleotide, the second primer extension product may itself serve as a template for the synthesis of the first primer extension product.

Furthermore, the second primer oligonucleotide may serve as the initiator of the synthesis of the second primer extension product starting from the starting nucleic acid chain to begin the amplification. In one embodiment, the sequence of the second primer is completely complementary to the corresponding sequence segment of a starting nucleic acid sequence. In a further embodiment, the sequence of the second primer oligonucleotide is only partially complementary to the corresponding sequence segment of a starting nucleic acid chain. However, this aberrant complementarity is not intended to prevent the second primer oligonucleotide from starting a predominantly sequence-specific primer extension reaction. The respective differences in complementarity of the second primer oligonucleotide to the respective position in the starting nucleic acid chain are preferably in the 5 'segment of the second primer oligonucleotide, so that predominantly complementary base pairing and initiation of the synthesis is possible in the 3' segment. For the initiation of the synthesis, for example, especially the first 4-10 positions in the 3 'segment should be fully complementary to the template (starting nucleic acid chain). The remaining nucleotide positions may differ from perfect complementarity. Thus, the extent of perfect complementarity in the 5 'segment can range between 10% to 100%, more preferably between 30% and 100% of the base composition. Depending on the length of the second primer oligonucleotide, this deviation from complete complementarity in the 5 'segment will range from 1 to 40, more preferably 1 to 20 nucleotide positions. In another embodiment, the second primer oligonucleotide binds only to its 3 'segment to the starting nucleic acid chain but not to its 5' segment. The length of such a 3'-segment of the second primer oligonucleotide fully complementary to start nucleic acid chain comprises regions between 6 and 40 nucleotides, better between 6 and 30 nucleotides, preferably between 6 and 20. The length of a corresponding, not to start nucleic acid chain complementary 5 'segment of the second primer oligonucleotide includes regions between 5 and 60, more preferably between 10 and 40 nucleotides. The second primer oligonucleotide may thus initiate synthesis from a starting nucleic acid chain. In a subsequent synthesis of the first primer extension product, sequence portions of the second primer oligonucleotide are copied from polymerase so that again in subsequent synthesis cycles a fully complementary primer binding site is formed within the first primer extension product for binding of the second primer oligonucleotide and is available in subsequent synthetic cycles.

In another embodiment, the second primer oligonucleotide may be used in the preparation of a starting nucleic acid chain. In this case, such a second primer oligonucleotide to a nucleic acid (eg a single-stranded genomic DNA or RNA or its equivalents comprising a target sequence) predominantly / preferably sequence-specifically bind and initiate a template-dependent primer extension reaction in the presence of a polymerase. The binding position is chosen such that the primer extension product comprises a desired target sequence. The extension of the second primer oligonucleotide results in a strand which has template-complementary sequence. Such a strand can be detached from the template (eg by heat or alkali) and thus converted into a single-stranded form. Such a single-stranded nucleic acid chain can serve as a starting nucleic acid chain at the beginning of the amplification. Such a start nucleic acid chain comprises in its 5 'segment the sequence portions of the second primer oligonucleotide, furthermore it comprises a target sequence or its equivalents and a primer binding site for the first primer oligonucleotide. Further steps are explained in the section "Starting nucleic acid chain".

In a preferred embodiment, the second primer oligonucleotide comprises at least in its 3 'segment sequence portions which can bind complementarily and sequence specifically to a sequence segment of a target sequence and initiate / support a successful primer extension reaction by polymerase. The length of such a sequence segment includes regions of 6 and 40 nucleotides, more preferably 8 to 30 nucleotides, preferably 10 to 25 nucleotides.

In one embodiment, the second primer oligonucleotide comprises copyable sequence segments in its 3 'and 5' segments, which are copied upon synthesis of the first primer extension product of polymerase. Thus, all sequence sections of the second primer are copied from polymerase. This leads to formation of a primer binding site in the 3 'segment of the first primer extension product.

In one embodiment, the second primer oligonucleotide, with its copatible portions in length, corresponds to the 3 'segment of the first primer extension product that is not bound by the controller oligonucleotide. In the complex comprising the second primer oligonucleotide and the first primer extension product, the 3 'end of such a second primer oligonucleotide is adjacent to the controller oligonucleotide bound to the first primer extension product. Extension of such a primer is done using the first primer extension product as a template. When such a primer is extended, the displacement of the controller oligonucleotide from binding with the first primer extension product occurs via polymase-dependent strand displacement.

In another embodiment, the second primer oligonucleotide with its copiable sequence portions is shorter than the 3 'segment of the first primer extension product that is not bound by the controller oligonucleotide. Thus, in the complex comprising the second primer oligonucleotide and the first primer extension product, there is a single-stranded portion of the first primer extension product between the 3 'end of such primer and the controller oligonucleotide bound to the first primer extension product. Extension of such a primer is done using the first primer extension product as a template. When such a primer is extended, the displacement of the controller oligonucleotide from binding with the first primer extension product occurs by polymase-dependent strand displacement.

In another embodiment, the second primer oligonucleotide, with its copatible portions, is longer than the 3 'segment of the first primer extension product which is not bound by the controller oligonucleotide. In the complex of the second primer oligonucleotide and the first primer extension product, the 3 'segment of the second primer and the 5' segment of the controller oligonucleotide compete for binding to the first primer extension product. The binding of the 3 'segment of the second primer to the first primer extension product required for initiation of the synthesis takes place with simultaneous partial displacement of the 5' segment of the controller oligonucleotide.

Upon initiation of the synthesis by polymerase, extension of such a primer is performed using the first primer extension product as a template. When such a primer is extended, the displacement of the controller oligonucleotide from binding with the first primer extension product occurs via polymase-dependent strand displacement. The sequence length of the 3 'segment of the second primer oligonucleotide displacing the 5' segment of the controller oligonucleotide may comprise the following ranges: 1 to 50 nucleotides, more preferably 3 to 30 nucleotides, preferably 5 to 20 nucleotides. Use of second primer oligonucleotides of greater length, which exceeds the length of the 3 'segment of the first primer extension product, is advantageous, for example, in some embodiments. Such embodiments include, for example, a first primer extension product having a 3 'segment that is not bound by the controller oligonucleotide, having a length of 5 to 40 Nucleotides, better from 10 to 30 nucleotides. Especially with shorter 3 'segments, a longer second primer oligonucleotide provides improved sequence specificity upon initiation of synthesis.

The binding strength of the second primer oligonucleotide to its primer binding site depends on the length of the primer. In general, longer second primer oligonucleotides can be used at higher reaction temperatures.

Preferably, sequences of the first, second primer oligonucleotide and the controller oligonucleotide are matched to one another such that side reactions, e.g. Primer-dimer formation, minimized. For this purpose, for example, the sequence of the first and second primer oligonucleotides are adapted to each other such that both primer oligonucleotides are incapable of undergoing an amplification reaction in the absence of an appropriate template and / or target sequence and / or starting sequence. Start nucleic acid chain. This can be achieved, for example, in that the second primer oligonucleotide does not comprise a primer binding site for the first primer oligonucleotide and the first primer oligonucleotide does not comprise a primer binding site for the second primer oligonucleotide. Furthermore, it should be avoided that the primer sequences comprise extended self-complementary structures (self-complement).

The synthesis of the second primer extension product is a primer extension reaction and forms a partial step in the amplification. The reaction conditions during this step are adjusted accordingly. The reaction temperature and the reaction time are chosen so that the reaction can take place successfully. The particular preferred temperature in this step depends on the polymerase used and the binding strength of the respective second primer oligonucleotide to its primer binding site and includes, for example, ranges from 15 ° C to 75 ° C, more preferably from 20 to 65 ° C, preferably from 25 ° C to 65 ° C. The concentration of the second primer oligonucleotide comprises ranges from 0.01 μmol / L to 50 μmol / L, more preferably from 0.1 μmol / L to 20 μmol / L, preferably from 0.1 μmol / L to 10 μmol / L.

In one embodiment, all steps of the amplification run under stringent conditions which prevent or slow down formation of nonspecific products / by-products. Such conditions include, for example, higher temperatures, for example above 50 ° C.

If more than one specific nucleic acid chain has to be amplified in one batch, in one embodiment preferably sequence-specific primer oligonucleotides are used for amplification of corresponding respective target sequences.

In one embodiment, the synthesis of the first and second primer extension products proceeds at the same temperature. In another embodiment, the synthesis of the first and second primer extension products proceeds at different temperatures. In another embodiment, synthesis of the second primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature. In another embodiment, synthesis of the second primer extension product and strand displacement by controller oligonucleotide proceeds at different temperatures.

polymerases:

Preferably, template-dependent polymerases are used which are capable of strand displacement.

For example, in particular embodiments, a large fragment of Bst polymerase or its modifications (e.g., Bst 2.0 DNA polymerase) can be used. Furthermore, Klenow fragment, Vent exo minus polymerase, Deepvent exo minus DNA polymerase, large fragment of Bsu DNA polymerase, large fragment of Bsm DNA polymerase can be used. Vent exo minus polymerase, Deepvent exo minus DNA (from NEB), and PyroPhage polymerase from Lucigen are dinostable enzymes with strand displacement activity.

In one embodiment, polymerases are used which exhibit no activity at room temperature, so-called hot-start polymerases or warm-start polymerases. An example of this is provided by Bst 2.0 Polymerase Warm Start (from NEB).

Preferred Embodiments of the Third Primer Oligonucleotide

(Component of the second amplification system)

In one embodiment, the third primer oligonucleotide is identical to the first primer oligonucleotide of the first amplification system.

In another embodiment, the third primer oligonucleotide is not identical to the first primer oligonucleotide of the first amplification system.

The length of the third primer oligonucleotide may be between 15 and 100 nucleotides, preferably between 20 and 60 nucleotides, more preferably between 30 and 50 nucleotides. The CG content is for example between 20% and 80%, better between 30 and 79%. The nucleotide building blocks are preferably linked to one another via customary 5'-3 'phosphodiester bond or phosphothioester bond. Such a primer oligonucleotide can be chemically synthesized in the desired form.

• • natürliche Nukleotide (dA, dT, dC, dG etc.) oder deren Modifikationen ohne veränderte Basen-Paarung
• • Modifizierte Nukleotide, nuklease-resistente Phosphorothioat-Verbindungen (PTO) Modifikationen, LNA-Modifikationen, 2-Amino-dA, 2-thio-dT oder andere Nukleotid-Modifikationen mit abweichender Basen-Paarung (z.B. Universal-Basenpaaren, wie beispielsweise Inosine 5-Nitroindol).

In one embodiment, the third primer oligonucleotide may include nucleotide monomers that do not or only insignificantly affect the function of the polymerase, including, for example:

• • natural nucleotides (dA, dT, dC, dG etc.) or their modifications without altered base pairing
• Modified nucleotides, nuclease-resistant phosphorothioate compounds (PTO) modifications, LNA modifications, 2-amino-dA, 2-thio-dT or other nucleotide modifications with deviant base pairing (eg, universal base pairs, such as inosine 5 -Nitroindol).

In a preferred embodiment, the 3'-OH end of the third primer oligonucleotide is preferably free of modification and has a functional 3'-OH group which can be recognized by polymerase and extended template-dependent. In a further preferred embodiment, the 3 'segment of the third primer comprises at least one phosphorothioate compound such that no degradation of 3' end of the primers can occur by 3 'exonuclease activity of polymerases.

In another embodiment, the 3 'end of the third primer is blocked. For example, with a 3'-phosphate group or with a C3 linker. Activation of the 3 'segment of the third primer oligonucleotide in these embodiments occurs only in the second amplification, e.g. using 3'-5 'exonuclease activity of the second polymerase.

The third primer oligonucleotide preferably does not comprise any sequence segments which can bind complementarily to the first region of the controller oligonucleotide.

The third primer oligonucleotide ( 21 - 40 ) may be in different arrangements relative to the first amplification fragment 1.1 (the first amplification product 1.1 comprising a target sequence).

Thus, the second amplification reaction using a first amplification fragment 1.1 as template (starting nucleic acid chain 2.1 ), the third and fourth primers must bind within sequences dictated by the first amplification fragment and be extended by polymerase.

In a preferred embodiment, the third primer oligonucleotide may be attached to a strand of the first amplification fragment 1.1 tie. Preferably, the third primer oligonucleotide binds to the second primer extension product and can thereby initiate primer extension using a suitable polymerase and reaction conditions.

In one embodiment, this binding of the dtag primer oligonucleotide is preferably carried out in the 3 'segment of the second primer extension product.

The third primer oligonucleotide preferably comprises a sequence segment in its 3 'region which can bind complementarily or predominantly complementarily to the second primer extension product under used reaction conditions such that polymerase is capable of directing the synthesis of the third primer extension product initiate.

The length of this segment preferably comprises regions between 6 and 30 nucleotides, more preferably between 8 and 25 nucleotides, preferably between 10 and 20 nucleotides, more preferably between 10 and 15 nucleotides. In one embodiment, this segment is fully complementary to the corresponding segment of the second primer extension product. In a further embodiment, this segment comprises at least one mismatch to the primer extension product. Preferably, the position of this mismatch is no closer than -4 position relative to the 3 'end of the third primer, better not closer than -5, even better not closer than -6, more preferably no closer than position -8 with respect to the 3' End of the third primer oligonucleotide. Since the third primer oligonucleotide can also bind to the controller oligonucleotide with this 3 'segment, binding of such a mismatch of this segment to the controller oligonucleotide is weakened. Thus, in the choice of length and number of mismatches, it is contemplated that while the 3 'segment is intended to bind to the second primer extension product and initiate a primer extension reaction, it is also noted that this segment is functional at the reaction conditions used does not irreversibly bind to the controller oligonucleotide and thus inactivate it.

The choice of position and length thus takes into account interactions between the third primer oligonucleotide and the controller, as well as the second primer extension product.

The position of the binding of this segment to the second primer extension product can be chosen differently ( 28 - 39 ).

For example, the third primer oligonucleotide may bind predominantly complementary to the same sequence segment as the first primer oligonucleotide. The position of the 3 'end of the third primer oligonucleotide matches the position of the 3' end of the first primer oligonucleotide. Thus, the third primer oligonucleotide may at least partially (with its 3 'segment) bind complementarily to a portion of the target sequence. The 3 'end of the third primer is thus within the second blocking unit of the controller oligonucleotide and can not be extended by polymerase using the template as template ( 29 ).

In another embodiment, the third primer oligonucleotide binds predominantly complementary to the second primer extension product, with its 3 'end being shifted with respect to the 3' end of the first primer oligonucleotide. In one embodiment, the 3 'end of the third primer oligonucleotide is shifted by at least one nucleotide in the 3' direction of the second primer extension product. Thus, the segment of the second primer extension product to which the third primer oligonucleotide is capable of predominantly complementary binding is displaced in the 3 'direction ( 28 ).

In another embodiment, the 3 'end of the third primer oligonucleotide is shifted by at least one nucleotide in the 5' direction of the second primer extension product. Thus, the segment of the second primer extension product, to which the third primer oligonucleotide can bind predominantly complementarily, is shifted in the 5 'direction ( 30 )

In another embodiment, the binding position of the third primer oligonucleotide is shifted in the 5 'direction of the second primer extension product so that it does not overlap with the binding position of the first primer oligonucleotide of the first amplification system. Thus, the segment of the second primer extension product to which the third primer oligonucleotide is capable of predominantly complementary binding is shifted in the 5 'direction relative to the binding site of the first primer oligonucleotide ( 31 )

The third primer oligonucleotide thus comprises sequence segments which can bind complementarily to the controller oligonucleotide. The segment of the controller oligonucleotide which can bind complementary to the third primer oligonucleotide are called the fourth region of the controller oligonucleotide. To prevent unwanted primer extension of the third primer oligonucleotide on the controller, at least positions of the controller oligonucleotide located around the 3 'end of the third primer oligonucleotide are modified. For example, this can be done in a similar way as in Modifkationen the second Blockung unit. The segment of the controller comprising such modifications (which prevent extension of the third primer oligonucleotide) is referred to as the fourth blocking unit ( 28 - 31 ). Their position depends on the potential binding position of the third primer oligonucleotide within the controller.

The third primer oligonucleotide should be able to be copied in reverse synthesis and also serves as a template for the synthesis of the fourth primer extension product.

The third primer oligonucleotide, in one embodiment, comprises at least one sequence segment in its 3 'region which can complementarily bind complementarily to the first target sequence predominantly complementary. This segment can be predominantly complementary to the second primer extension product (comprising corresponding segments of the target sequence), which polymerase can perform a primer extension reaction.

In a further embodiment, the third primer oligonucleotide comprises at least one sequence segment in its 5 'region which is not complementary to the target sequence. This segment may, for example, comprise from 1 to 60 nucleotides and may serve, for example, for other purposes, for example barcoding, cloning, immobilization, probe binding, etc. The sequence composition of this 5 'region is adapted so as not to interfere with the second amplification.

The third primer oligonucleotide may comprise further modifications, eg fluorescent dyes (eg FAM, Cy5 etc.), fluorescence quenchers (eg BHQ1, BHQ2 etc), affinity tags (eg biotin, digoxigenin etc.). In a wet embodiment, the third primer oligonucleotide can be inserted into linkers (eg C3 or HEG linker) such that its 5 'region of polymerase remains uncopied.

In one embodiment, the third primer is immobilized on a solid phase prior to the second amplification reaction. The primer extension of this third primer oligonucleotide thus results in immobilization of the entire third primer extension product.

The concentration of the third primer oligonucleotide used may, for example, comprise ranges between 0.01 μmol / l to about 10 μmol / l, more preferably between 0.1 μmol / l and about 2 μmol / l.

Preferred Embodiments of the Fourth Primer Oligonucleotide

(Component of the second amplification system)

In one embodiment, the fourth primer oligonucleotide is identical to the second primer oligonucleotide of the first amplification system.

In another embodiment, the fourth primer oligonucleotide is not identical to the second primer oligonucleotide of the first amplification system.

The length of the fourth primer oligonucleotide may be between 15 and 100 nucleotides, preferably between 20 and 60 nucleotides, more preferably between 30 and 50 nucleotides. The CG content is for example between 20% and 80%, better between 30 and 79%. The nucleotide building blocks are preferably linked to one another via customary 5'-3 'phosphodiester bond or phosphothioester bond. Such a primer oligonucleotide can be chemically synthesized in the desired form.

• • natürliche Nukleotide (dA, dT, dC, dG etc.) oder deren Modifikationen ohne veränderte Basen-Paarung
• • Modifizierte Nukleotide, nuklease-resistente Phosphorothioat-Verbindungen (PTO) Modifikationen, LNA-Modifikationen, 2-Amino-dA, 2-thio-dT oder andere Nukleotid-Modifikationen mit abweichender Basen-Paarung (z.B. Universal-Basenpaaren, wie beispielsweise Inosine 5-Nitroindol).

In one embodiment, the fourth primer oligonucleotide may include nucleotide monomers that do not or only insignificantly affect the function of the polymerase, including, for example:

• • natural nucleotides (dA, dT, dC, dG etc.) or their modifications without altered base pairing
• Modified nucleotides, nuclease-resistant phosphorothioate compounds (PTO) modifications, LNA modifications, 2-amino-dA, 2-thio-dT or other nucleotide modifications with deviant base pairing (eg, universal base pairs, such as inosine 5 -Nitroindol).

In a preferred embodiment, the 3'-OH end of the fourth primer oligonucleotide is preferably free of modification and has a functional 3'-OH group which can be recognized by polymerase and extended template-dependent. In a further preferred embodiment, the 3 'segment of the fourth primer comprises at least one phosphorothioate compound so that no degradation of 3'-end of the primers by 3'-exonuclease activity of polymerases can take place.

In another embodiment, the 3'-end of the fourth primer is blocked. For example, with a 3'-phosphate group or with a C3 Linker. The activation of the 3 'segment of the fourth primer oligonucleotide in these embodiments takes place only in the second amplification, for example using 3'-5' exonuclease activity of the second polymerase.

The fourth primer oligonucleotide preferably does not include any sequence segments which are capable of complementary binding to the first region of the controller oligonucleotide.

The fourth primer oligonucleotide ( 24 - 40 ) may be in different arrangements relative to the first amplification fragment 1.1 (the first amplification product 1.1 comprising a target sequence).

Thus, the second amplification reaction using a first amplification fragment 1.1 as template (starting nucleic acid chain 2.1 ), the third and fourth primers must bind within sequences dictated by the first amplification fragment and be extended by polymerase.

In a preferred embodiment, the fourth primer oligonucleotide may be attached to a strand of the first amplification fragment 1.1 tie. Preferably, the fourth primer oligonucleotide binds to the first primer extension product and can thereby initiate primer extension using a suitable polymerase and reaction conditions.

This binding of the fourth primer oligonucleotide is preferably carried out in one embodiment in the 3 'segment of the first primer extension product.

The fourth primer oligonucleotide preferably comprises a sequence segment in its 3 'region which can bind complementarily or predominantly complementarily to the first primer extension product under used reaction conditions such that polymerase is capable of directing the synthesis of the fourth primer extension product initiate.

The length of this segment preferably comprises regions between 6 and 40 nucleotides, more preferably between 8 and 30 nucleotides, preferably between 10 and 25 nucleotides, particularly preferably between 10 and 20 nucleotides. In one embodiment, this segment is fully complementary to the corresponding segment of the first primer extension product. In a further embodiment, this segment comprises at least one mismatch to the first primer extension product. Preferably, the position of this mismatch is no closer than -4 position with respect to the 3 'end of the fourth primer, better not closer than -5, even better not closer than - 6, more preferably not closer than position -8 with respect to the 3' End of the fourth primer oligonucleotide.

The position of the binding of this segment to the first primer extension product can be chosen differently ( 32 - 40 ).

For example, the fourth primer oligonucleotide may bind predominantly complementary to the same sequence segment as the second primer oligonucleotide. The position of the 3 'end of the fourth primer oligonucleotide coincides with the position of the 3' end of the second primer oligonucleotide. Thus, the fourth primer oligonucleotide comprises at least partially (with its 3 'segment) a portion of the target sequence.

In a further embodiment, the fourth primer oligonucleotide binds predominantly complementary to the first primer extension product, its 3 'end being shifted with respect to the 3' end of the second primer oligonucleotide. In one embodiment, the 3 'end of the fourth primer oligonucleotide is shifted by at least one nucleotide in the 3' direction of the first primer extension product. Thus, the segment of the first primer extension product to which the fourth primer oligonucleotide is capable of predominantly complementary binding is displaced in the 3 'direction ( 1 ).

In another embodiment, the 3 'end of the fourth primer oligonucleotide is shifted by at least one nucleotide in the 5' direction of the first primer extension product. Thus, the segment of the first primer extension product to which the fourth primer oligonucleotide is capable of predominantly complementary binding is shifted in the 5 'direction.

The fourth primer oligonucleotide preferably does not comprise sequence segments which are capable of complementary binding to the controller oligonucleotide.

The fourth primer oligonucleotide is said to be capable of being copied upon reverse synthesis and also serves as a template for synthesis of the third primer extension product.

The fourth primer oligonucleotide in one embodiment comprises at least one sequence segment in its 3 'region which comprises portions of the first target sequence and thus can bind to a strand which is complementary to the target sequence. This segment can be predominantly complementary to the first primer extension product (comprising corresponding complementary segments to the target sequence), which polymerase can perform a primer extension reaction.

In a further embodiment, the fourth primer oligonucleotide comprises at least one sequence segment in its 5 'region, which does not comprise any sequence segments of the first target sequence. This segment may, for example, comprise from 1 to 60 nucleotides and may serve, for example, for other purposes, for example barcoding, cloning, immobilization, probe binding, etc. The sequence composition of this 5 'region is adapted so as not to interfere with the second amplification.

The fourth primer oligonucleotide may include further modifications, eg, fluorescent dyes (eg, FAM, Cy5, etc.), fluorescence quenchers (eg, BHQ1, BHQ2, etc.), affinity tags (eg, biotin, digoxigenin, etc.) In a wet embodiment, the fourth Primer oligonucleotide in linker (eg C3 or HEG linker) such that its 5 'region of polymerase remains uncopied.

In one embodiment, the fourth primer is immobilized on a solid phase prior to the second amplification reaction. The primer extension of such a fourth primer oligonucleotide thus results in immobilization of the entire fourth primer extension product.

The concentration of the fourth primer oligonucleotide used may comprise, for example, ranges between 0.01 μmol / l to about 10 μmol / l, more preferably between 0.1 μmol / l and about 2 μmol / l.

Polymerases of the second amplification system:

Many polymerases are known for carrying out a PCR.

In one embodiment, thermostable template-dependent DNA polymerases are used which have a 5'-3 'exonuclease activity.

In one embodiment, Taq polymerase and / or its formulations and / or their modifications (e.g., Ampli-Taq) are used to carry out the second amplification.

In a further embodiment, thermostable template-dependent DNA polymerases are used which have a strand-displacement activity. For example, Vent Exo minus (from NEB), PyroPhage Polymerase (from Lucigene), SD Polymerase (from Bioron).

In another embodiment, thermostable template-dependent DNA polymerases are used which have a 3'-5 'proof reading activity. For example, Vent exo plus, Deep Vent exo plus (from NEB), Pfu Polymerase (Jena Biosciences), Phusion Polymerase (NEB).

In another embodiment, thermostable template-dependent DNA polymerases are used which are conjugated to another protein, for example, to increase the polymerase's processivity. For example, Phusion Polymerase (NEB).

Preferred embodiments of the detection system

The detection system comprises at least one fluorescent reporter and at least one oligonucleotide probe which is capable of hybridizing to at least one of the primer extension product formed during amplification. Furthermore, a detection system may comprise a fluorescent quencher (called a quencher) which matches the fluorescent reporter, so that this quencher is able to reduce the fluorescence signals of the fluorescent reporter under certain circumstances or to reduce the signal intensity. Furthermore, a detection system may include a fluorescent reporter-appropriate donor fluorophore such that this donor fluorophore is capable of enabling the fluorescence signals of the fluorescent reporter in certain circumstances by energy transfer. Furthermore, the detection system may comprise the activator oligonucleotide, such activator oligonucleotide comprising either a fluorescent reporter or a donor fluorophore or a fluorescence quencher.

The arrangement of individual elements (fluorescent reporter, fluorescence quencher, donor fluorophore) on the oligonucleotide probe and / or on the controller oligonucleotide to lead in the presence of a primer extension product to form respective complementary complexes to change the fluorescence signal from the fluorescent reporter.

A person skilled in the art knows a large number of reporter systems which have been developed in the realm of real-time PCR in the last 20 years. These include, for example, probes with self-complementary segments, for example: so-called "molecular beacons", exonuclease-degradation-based probes (so-called "Taqman" probes which are specifically cleaved using Taq polymerase 5'-3 'nuclease activity), probe probes Systems with two oligonucleotides which are labeled with FRET pair and can be arranged in a strand so that signal is generated; primer-based probes (eg LUX primer, which change the intensity of the signal when self-complementary structures unfold during reverse synthesis), "Scorpion primer" (with segments complementary to strand synthesized before the primer), etc. Some probes can be used as end point measurement in signal acquisition (eg, molecular beacons). Some probes are used to detect the kinetics of PCR, e.g. 5'-3'-nuclease probes. Many different arrangements of probes and fluorescent reporters, fluorescence quenchers and donor fluorophores are known in the literature. Also, many fluorescent reporter quenchers as well as fluorescent reporter donor fluorophores (also called donor-acceptor pairs or also called FRET pairs) are known ("Fluorescent Energy Transfer Nucleic Acid Probes" Ed Didenko, 2006, for example, Chapter 1 and 2; Product Description "Flurescent Molecular Probes", published by Gene Link Inc.). Most probes have been developed for PCR-based procedures, with both specific arrangement of primers, probes, and polymerases used, e.g. with 3'-exonuclease (FEN).

In general, such probes may bind more or less specifically to the DNA fragments generated during amplification, the signal being altered as a result of such binding alone or in combination with other events (eg, nuclease degradation or attachment of another oligonucleotide to adjacent segments) , This change can be detected and quantified.

Because of the use of the PCR in the second amplification, therefore, also such known techniques and probes can be used. The sequences of oligonucleotide probes are adapted to the amplification products formed during the reaction.

The choice of a probe also depends on whether, for example, a particular variant of the target sequence has to be detected by means of a specific sequence-specific probe or should, for example, only consumption of primers (as a sign of amplification) be registered. Different probe formats can thus be selected, depending on the task.

In the following, therefore, the special features of such an adaptation for some embodiments will be discussed in the first place.

Hoterogenic format

In this case, components of both amplification systems are separated at the beginning of the first amplification, so that no influence of oligonucleotide probes or PCR primers on the first amplification can take place. Conversely, upon completion of the first amplification, an aliquot is transferred from the first amplification to the second amplification. The resulting dilution of reaction components of the first amplification system favors the use of known for a PCR reaction probe constructions. For example, in the presence of the controller oligonucleotide in concentrations of below 100 nmol / l, better below 10 nmol / l, preferably below 1 nmol / l, more preferably below 0.01 nmol / l, the probe binding to complementary segments of the formed primer extension fragments under PCR conditions hardly affected, so that well-known probe-based detection methods can be applied to a person skilled in the art.

The oligonucleotide probes may bind to one of the formed primer extension products (eg to the third and fourth primer extension product) and this binding event may be detected by various techniques (eg, using Taqman probes and a Taq polymerase or by binding from "Molecular Beacons" to complementary segments of corresponding primer extension products.

Homogeneous format

• • Anwesenheit von Controller-Oligonukleotide kann die Bindung von Oligonukleotid-Sonden an die gebildeten Primer-Verlängerungsprodukte beeinflussen. Das ist beispielsweise dann der Fall, wenn eine Sonde und ein Controller signifikante Überlappungen in Segmenten umfassen, welche komplementär zu Primer-Verlängerungsprodukten sind (z.B. dritter Bereich des Controllers und Oligonukleotid-Sonde können teilweise ein das gleiche Segment des ersten und / oder des dritten Primer-Verlängerungsproduktes hybridisieren). Aus diesem Grund ist es vorteilhaft, das Ausmass solcher Überlappungen zu begrenzen. Dafür kann beispielsweise die Länge von Segmenten bei Controller und Sonde, dermassen angepasst werden, dass diese möglichst geringe Überlappung aufweisen. Vorzugsweise überlappen die Sequenzsegmente bei komplementärer Bindung an die gebildeten Primer-Verlängerungsprodukte nicht.
• • Oligonukleotid-Sonden können an das Controller-Oligonukleotid ggf. binden und diesen bei Strangverdrängung beeinflussen. Das kann beispielsweise dann erfolgen, wenn das Controller-Oligonukleotid und die Oligonukleotid-Sonde je ein Segment umfassen, welches zu Ausbildung eines doppelsträngigen Kompleses zwischen dem Controller und der Sonde führt. Aus diesem Grund ist es vorteilhaft, das Ausmass solcher komplementären Segmente zu begrenzen. Dafür kann beispielsweise die Länge von Segmenten bei Controller und Sonde, dermassen angepasst werden, dass diese möglichst geringe Komplementarität aufweisen. Vorzugsweise umfassen Controller und der Sonde keine komplementären Sequenzsegmente.

In a homogeneous format, the components of both amplification systems are in the same approach at the beginning of the first amplification and can therefore interact with other components and intervene in certain processes. In particular, the presence of effective concentrations of controller oligonucleotides (eg between 0.01 μmol / L and 10 μmol / L) and oligonucleotide probes (eg between 0.01 μmol / L and 10 μmol / L) can lead to interactions between these components could influence sub-steps or results of these steps. For this reason, the following aspects, among others, have to be taken into account:

• Presence of controller oligonucleotides may affect the binding of oligonucleotide probes to the primer extension products formed. This is the case, for example, when a probe and a controller include significant overlaps in segments that are complementary to primer extension products (eg, third region of the controller and oligonucleotide probe may be partially the same segment of the first and / or third primer Hybridize extension product). For this reason, it is advantageous to limit the extent of such overlaps. For example, the length of segments in the case of the controller and probe can be adjusted to such an extent that they have the least possible overlap. Preferably, the sequence segments do not overlap upon complementary binding to the formed primer extension products.
• • Oligonucleotide probes may bind to the controller oligonucleotide and affect it upon strand displacement. This can be done, for example, when the controller oligonucleotide and the oligonucleotide probe each comprise a segment which results in the formation of a double-stranded complement between the controller and the probe. For this reason, it is advantageous to limit the extent of such complementary segments. For example, the length of segments in the case of the controller and probe can be adjusted to such an extent that they have as little complementarity as possible. Preferably, the controller and the probe do not include complementary sequence segments.

For such reasons, for example, in a homogeneous format, it is advantageous to make the oligonucleotide probe hybridize preferentially to the 3 'segment of the first or third primer extension product. These segments are not hybridized by the controller oligonucleotide.

Another essential aspect of this invention is a possible polymerase change upon switching from the first to the second amplification: the first polymerase can be inactivated and the second polymerase can be activated. This makes it possible, for example, to use 5'-3'-nuclease-sensitive probes ("Taqman probes" during the second amplification: in the first amplification, for example, Bst-Polymerase Large Fragment is preferably used.) This fragment can not cleave the Taqman probes. At the beginning of the second amplification, the Bst polymerase Large fragment is inactivated and the Taq polymerase (eg used as a hot-start polymerase) is activated thereby allowing the 5'-3'-nuclease-sensitive probes to be used.

Depending on the chosen constellation between a fluorescent reporter and / or a donor fluorophore and / or a fluorescence quencher, this change can lead to an increase or decrease in the signal intensity of the fluprescent repeater. This change can be detected by known suitable means (e.g., in a real-time PCR device such as StepOne PCR or Lightcycler or Rotorgene, see data from manufacturers) during or after an expired reaction. The detection of the changes in the signal can allow conclusions to be drawn about the course of the reaction: e.g. Amplitude of the signal, kinetics, time or concentration dependence of the signal appearance. When using multiple target sequences, multiplex analyzes can be coded according to different spectral properties, so that several reactions can be observed parallel to each other.

The present invention describes some embodiments of oligonucleotide probes which are particularly advantageous for detecting the progress of the reaction.

The oligonucleotide probe is an oligonucleotide, which is preferably constructed from DNA nucleotides. In another embodiment, this oligonucleotide can be constructed from other nucleotide monomers, for example from RNA or from nucleotide modifications, for example with sugar-phosphate backbone modifications such as PTO or LNA or 2'-O-Me. In another embodiment, this oligonucleotide sample is a mixed polymer comprising both DNA and non-DNA elements (eg RNA, PTO LNA). This oligonucleotide probe may comprise further oligonucleotide modifications, eg linkers or spacers (eg C3 , HEG, Abasic monomers, eg THF modification).

The base composition of the oligonucleotide probe preferably comprises bases which can undergo complementary binding with natural nucleobases (A, C, T, G) under hybridization conditions. In a further embodiment, the probe also includes modifications comprising, for example, universal bases (eg, inosine, 5-nitro-indole). The probe may include other modifications that affect binding behavior of oligonucleotides, eg MGB modifications.

The length of the oligonucleotide probe is preferably between 8 and 80 nucleotides, more preferably between 12 and 80 nucleotides, more preferably between 12 and 50, preferably between 12 and 35 nucleotides. The oligonucleotide probe typically comprises a segment which can complementarily bind complementarily to at least one of the formed products. In one embodiment, the oligonucleotide probe comprises at least one further segment which can not bind to one of the formed primer extension products in a complementary manner.

The probe may be arranged differently in realization to other components of the amplification systems. Certain embodiments are preferred:

In one embodiment, an oligonucleotide probe comprises at least one of the formed primer extension products ( P1.1 -ext, P2.1 -ext, P3.1 -ext, P4.1 -Ext) substantially complementary sequence segment which can hybridize to at least one of the primer extension product formed under suitable conditions (hybridization conditions). In a further embodiment, this sequence segment is complementary to the first and / or the third primer extension product. In another embodiment, this sequence segment is complementary to the first and / or the third primer extension product, wherein the oligonucleotide probe can bind complementarily in the 3 'segment of the respective primer extension product which is not complementarily bound by the controller oligonucleotide. In a further embodiment, this sequence segment is complementary to the second and / or the fourth primer extension product. In a further embodiment, this sequence segment of the oligonucleotide probe comprises a length which is in the range between 10 nucleotides and 50, more preferably between 15 and 40, preferably between 15 and 30 nucleotides. In another embodiment, this sequence segment of the oligonucleotide probe does not comprise a sequence region that is substantially complementary to the controller oligonucleotide. In another embodiment, this sequence segment of the oligonucleotide probe comprises a sequence region which is substantially complementary to the controller oligonucleotide, the length of this segment being less than 20 nucleotides, more preferably less than 15 nucleotides, preferably less than 10 nucleotides, most preferably less as 5 nucleotides comprising

In another embodiment, this sequence segment of the oligonucleotide probe does not comprise a sequence region that is substantially complementary to one of the primer oligonucleotides. In a further embodiment, this sequence segment of the oligonucleotide probe comprises a sequence region that is substantially complementary to one of the primer oligonucleotides, the length of this segment being less than 20 nucleotides, better less than 15 nucleotides, preferably less than 10 nucleotides, more preferably less than 5 nucleotides.

In another embodiment, this sequence segment of the oligonucleotide probe does not include a sequence region that is substantially identical to the sequence of the third region of the controller oligonucleotide. In another embodiment, this sequence segment of the oligonucleotide probe comprises a sequence region which is substantially identical to the sequence of the third region of the controller oligonucleotide, the length of this segment being less than 20 nucleotides, more preferably less than 15 nucleotides, preferably less than 10 nucleotides, more preferably less than 5 nucleotides,

In one embodiment, the controller oligonucleotide comprises one of the following components (a fluorescence reporter and / or a fluorescence quencher and / or a donor fluorophore) with at least one of these components located in the third region of the controller oligonucleotide.

In one embodiment, the oligonucleotide probe is at least partially cleavable by 5'-3'-nuclease. In another embodiment, the controller oligonucleotide is cleavable by 5'-3 'nuclease at least in its 5' segment (third region of the controller).

In another embodiment, an oligonucleotide probe comprises a sequence segment which is substantially complementary to the target sequence or its portion encompassed by one of the amplification products, the length of this segment being in the range of 5 to 50 nucleotides, more preferably 10 to 10 nucleotides 40, preferably between 15 and 30 nucleotides. In another embodiment For example, an oligonucleotide probe does not comprise a sequence segment substantially complementary to the target sequence or its complementary strand.

In one embodiment, the 3 'end of the oligonucleotide probe is blocked by a modification, eg, by a fluorophore or quencher or donor, or by another modification that prevents polymerase from using the oligonucleotide as a primer (eg, phosphate residue or C3 Linker or dideoxy nucleotide modification).

In another embodiment, the 3 'end of the oligonucleotide probe is free and can bind complementary to the first primer extension product and initiate synthesis by the polymerase. This allows the oligonucleotide probe to be extended like a primer, resulting in the formation of a probe extension product.

In one embodiment, the sequence composition of the probe corresponds to the sequence composition of the primer.

Position of fluorescent reporter, fluorescent quencher or donor fluorophore may vary depending on the embodiment of the oligonucleotide probe. These elements can be covalently bound to either one of the respective ends of the oligonucleotide probe or in the middle region. Many such modifications are known to one skilled in the art, for example, coupling of FAM reporter to the 3 'end or to the 5' end of a nucleotide, or use of dT-BHQ1 or dT-FAM or dT-TMR modifications for coupling of probes in Middle or inner sequence segment of the probe. Such modified oligonucleotide probes may be obtained from commercial suppliers (e.g., Sigma-Aldrich, Eurofins, IDT, Eurogentec, Thermofisher Scientific).

For example, the oligonucleotide probe comprises at least one sequence segment which is capable of undergoing substantially complementary binding with the third or fourth primer extension product formed during amplification under appropriate reaction conditions of a detection step. In this case, the oligonucleotide probe is bound for example to the single-stranded 3 'segment of the synthesized third primer extension product or to the synthesized 5' segment of the fourth primer extension product complementary. In this case, such a probe does not comprise sequence segments which are identical to or complementary to the controller. This will generate a complex comprising a primer extension product, oligonucleotide probe. The binding of the oligonucleotide probe and the controller oligonucleotide to the synthesized third primer extension product is preferably sequence-specific. The length of this sequence segment complementary to the 3 'segment of the first primer extension product is, for example, in the range of 8 to 80 nucleotides, more preferably 12 to 80 nucleotides, more preferably 12 to 50, preferably 12 to 35 nucleotides, more preferred between 15 and 25 nucleotides.

The detection system comprising at least one fluorescent reporter bound to either the oligonucleotide probe or the controller oligonucleotide is capable of generating the signal or fluorescence reporter signal intensity as a function of the binding of the oligonucleotide probe to change to complementary sequences. Depending on the embodiment of the detection system, this change may result in the generation and / or increase or decrease of the signal.

During amplification, the third and fourth primer extension products are separated such that 3 'segment of the third primer extension product and the corresponding fragment of the fourth primer extension product are in single stranded form. An oligonucleotide probe can bind to this 3 'segment of the third primer extension product or 5' segment of the fourth primer extension product during the reaction or only after its completion.

The binding is essentially sequence-specific, but can tolerate deviations from complete complementarity.

The binding of the oligonucleotide probe preferably does not prevent amplification. The concentration of the probe and its length are adjusted so that sufficient amount of primers can initiate the synthesis of the primer extension prodrugs.

As the reaction progresses, a sufficient amount of primer extension products are formed so that the oligonucleotide probe can also bind sufficiently to cause a detectable signal change.

A suitable detection system comprising at least one fluorescence reporter further comprises in one embodiment at least one fluorescent quencher suitable for the fluorescent reporter. The fluorescence quencher (also called a quencher) can function as a contact quencher or as a FRET quencher. Relevant examples in the literature are known ("Fluorescent Energy Transfer Nucleic Acid Probes" Ed Didenko, 2006, for example, Chapter 1 and 2; Product Description "Florescent Molecular Probes", published by Gene Link Inc.). For example, fluorescein (FAM) can serve as the fluorescent reporter, and BHQ-1 or BHQ-2 or TAMRA can occur as a suitable fluorescence quencher. In one embodiment, guanosine nucleobases may act as quenchers (e.g., in combination with a FAM as a reporter). Upon excitation of the fluorescent reporter with a suitable wavelength of light emission of the fluorescence signal occurs in the absence of the quencher. However, spatial proximity between the fluorescent reporter and a suitable quencher reduces / decreases the intensity of a fluorescent reporter. With increasing distance / separation of the fluorescent reporter and the quencher, the signal intensity increases.

Another suitable detection system comprising at least one fluorescence reporter further comprises, in one embodiment, at least one donor fluorophore (also called a donor) matching the fluorescent reporter so that a FRET pair is formed. Relevant examples in the literature are known ("Fluorescent Energy Transfer Nucleic Acid Probes" Ed Didenko, 2006, for example, Chapter 1 and 2, Product Description "Fluorescent Molecular Probes", published by Gene Link Inc.). A FRET pair usually includes a donor and an acceptor (usually a reporter acts as an acceptor). For example, as a fluorescent reporter tetramethylrodamine (TAMRA) can serve, as a suitable partner of a FRET pair FAM (donor) may occur, another example is FAM (donor) and Cy3 (as acceptor or reporter). When the donor excites (eg, FAM), the donor transfers the energy to the reporter (TAMRA or Cy3) so that the reporter himself is thereby enabled to use energy as electromagnetic radiation (detectable light signal or fluorescence signal). leave. This fluorescent signal of the reporter can be detected by suitable means. With increasing spatial separation of the reporter and the donor fluorophore, the signal decreases increasingly and is generally no longer measurable detectable from a distance of over 50 nucleotides (measured as 50 nucleotides of a double strand).

By appropriate positioning of individual elements of the detection system to oligonucleotide probe and / or activator oligonucleotide, it is possible to detect binding events of these components to the first primer extension product by signal increase or decrease.

Such detection may occur either during amplification (e.g., as an on-line detection) or at appropriate time intervals, or even at the end of a reaction.

Detection of the fluorescence signal takes place in a detection step. In the detection step of the method it should be checked whether the oligonucleotide probe is attached to the 3 'segment of the first primer extension product ( P1 -Ext) has bound complementarily or not. In the detection step, the reactor temperature is therefore adjusted so that the probe would be able to substantially complementarily bind to the 3 'segment of the first primer extension product. This temperature may either correspond to one of the temperatures during amplification or represent a step separate from amplification temperature steps.

During this step, the response from a light source can be excited with the light of a suitable wavelength. Depending on the design of the detection system, the wavelength is adapted to the absorption spectrum of the fluorescent reporter or of the donor fluorophore. If the oligonucleotide probe can bind to the 3 'end of a synthesized first primer extension product, a signal from the fluorescent reporter is expected. This signal has a characteristic spectrum of wavelengths and can be detected and quantified by a corresponding detection system. Current real-time PCR devices typically include both excitation light source and detection system for reporter fluorescence detection, as well as temperature-controlled reaction vessel containers. An example of this are real-time PCR devices such as StepOne or LightCycler or RotorGene.

The detection can be used to quantify one or more starting nucleic acid chains present in the reaction. Furthermore, the detection can be used to detect an availability of a starting nucleic acid chain at the beginning of a reaction. Furthermore, a detection system may be used in conjunction with an internal amplification control.

In another embodiment, two or more nucleic acid chains can be specifically amplified in an amplification approach. For example, combinations of specific first primers and / or specific activator oligonucleotides and / or specific second primers may be used. For example, target sequences and an internal amplification control are amplified in one approach.

It is therefore advantageous to carry out the detection of individual nucleic acid chains separately and independently of one another during their amplification.

In one embodiment, specific detection systems are used in each case, so that monitoring of the amplification of a nucleic acid chain by means of a specific detection system takes place. The specific signals of fluorescence reporters can be detected simultaneously. Preferably, the spectral properties of fluorescent reporters differ so much that they can be made by detecting respective fluorescence signals at characteristic wavelengths. For example, two or three or four fluorescent reporters can be used. The respective specific wavelengths of fluorescence signals are preferably more than 10 nm, better more than 20 nm, preferably more than 30 nm measured at maximum intensity (fluorescence peak) of a fluorescence spectrum). , Such combinations are known. For example, combinations comprising FAM and / or Cy3 and / or Cy5 or FAM and / or HEX and / or ROX are suitable. The respective quenchers are preferably chosen specifically for each fluorescent reporter, so that signal reduction by a quencher has a high efficiency. For example, combinations of FAM / BHQ-1 and HEX / BHQ-2 or FAM / BHQ-1 and Cy5 / BHQ-2 are used.

In a further embodiment, a detection system may be used which allows monitoring of the amplification of a group of different nucleic acid chains. This group may comprise two or more nucleic acid chains to be amplified. The components of a detection system are adjusted accordingly. In one embodiment, such a group of different nucleic acid chains to be amplified comprises, for example, at least one uniform sequence segment specific for the oligonucleotide probe used for complementary binding of an oligonucleotide probe under reaction conditions of a detection step. In a further embodiment, such a group of different nucleic acid ketones to be amplified comprises, for example, at least one sequence segment for a predominantly complementary binding of an oligonucleotide probe, the sequence composition of this sequence segment being different within this group. These differences may range from 1 to 10 nucleotides, preferably from 1 to 3 nucleotides.

Examples

Material and methods:

• • nicht modifizierte und modifizierte Oligonukleotide (Eurofins MWG, Eurogentec, Biomers, Trilink Technologies, IBA Solutions for Life Sciences)
• • Polymerasen NEB (New England Biolabs)
• • dNTP's: Jena Bioscience
• • interkallierender EvaGreen Farbstoff: Jena Bioscience
• • Puffer-Substanzen und andere Chemikalien: Sigma-Aldrich
• • Plastikware: Sarstedt

Reagents were purchased from the following commercial suppliers:

• Unmodified and modified oligonucleotides (Eurofins MWG, Eurogentec, Biomers, Trilink Technologies, IBA Solutions for Life Sciences)
• Polymerase NEB (New England Biolabs)
• • dNTP's: Jena Bioscience
• • intercalating EvaGreen dye: Jena Bioscience
• • Buffer substances and other chemicals: Sigma-Aldrich
• • Plastic goods: Sarstedt

• • Kalium Glutamat, 50 mmol/l, pH 8,0
• • Magnesium Acetat, 10 mmol/l
• • dNTP (dATP, dCTP, dTTP, dGTP), je 200 µmol/l
• • Polymerase (Bst 2.0 WarmStart, 120.000 U/ml NEB), 12 Units / 10 µl
• • Triton X-100, 0,1% (v/v)
• • EDTA, 0,1 mmol/l
• • TPAC (Tetrapropylammonium Chloride), 50 mmol/l, pH 8,0
• • EvaGreen Farbstoff (Farbstoff wurde entsprechend Anleitung des Herstellers in
• • Verdünnung 1:50 eingesetzt).
• •

solution 1 (Amplification reaction solution 1 ):

• • potassium glutamate, 50 mmol / l, pH 8.0
• • Magnesium acetate, 10 mmol / l
• • dNTP (dATP, dCTP, dTTP, dGTP), 200 μmol / l each
• • Polymerase (Bst 2.0 WarmStart, 120,000 U / ml NEB), 12 units / 10 μl
• • Triton X-100 , 0.1% (v / v)
• • EDTA, 0.1 mmol / l
• TPAC (tetrapropylammonium chloride), 50 mmol / l, pH 8.0
• • EvaGreen dye (dye was prepared according to manufacturer's instructions in
• • dilution 1:50 used).
• •

• • 1x Isothermal Puffer (New England Biolabs); in einfacher Konzentration enthält der Puffer:
  • 20 mM Tris-HCI
  • 10 mM (NH4)2SO4
  • 50 mM KCl
  • 2 mM MgSO4
  • 0.1% Tween® 20
  • pH 8.8@25°C)
• • dNTP (dATP, dCTP, dUTP, dGTP), je 200 µmol/l
• • EvaGreen Farbstoff (Farbstoff wurde entsprechend Anleitung des Herstellers in Verdünnung 1:50 eingesetzt)

solution 2 (Amplification reaction solution 2 ):

• 1x isothermal buffer (New England Biolabs); in simple concentration, the buffer contains:
  • 20 mM Tris-HCl
  • 10mM (NH4) 2SO4
  • 50 mM KCl
  • 2mM MgSO4
  • 0.1% Tween® 20
  • pH 8.8 @ 25 ° C)
• • dNTP (dATP, dCTP, dUTP, dGTP), 200 μmol / l each
• • EvaGreen dye (dye was used according to manufacturer's instructions in dilution 1:50)

• • 1x Isothermal Puffer (New England Biolabs); in einfacher Konzentration enthält der Puffer:
  • 20 mM Tris-HCI
  • 10 mM (NH4)2SO4
  • 50 mM KCl
  • 2 mM MgSO4
  • 0.1 % Tween® 20
  • pH 8.8@25°C)
• • dNTP (dATP, dCTP, dTTP, dGTP), je 200 µmol/l

solution 4 (Amplification reaction solution 4 ):

• 1x isothermal buffer (New England Biolabs); in simple concentration, the buffer contains:
  • 20 mM Tris-HCl
  • 10mM (NH4) 2SO4
  • 50 mM KCl
  • 2mM MgSO4
  • 0.1% Tween® 20
  • pH 8.8 @ 25 ° C)
• • dNTP (dATP, dCTP, dTTP, dGTP), 200 μmol / l each

All concentrations are indications of the final concentrations in the reaction. Deviations from the standard reaction are indicated accordingly.

The melting temperature (Tm) of the components involved became in solution at the concentration of 1 μmol / L of respective components 1 certainly. Different parameters are indicated in each case.

General information on reactions (first amplification)

The reaction was initiated by heating the reaction solutions to reaction temperature, since Bst 2.0 Polymerase warm start at lower temperatures is largely inhibited in their function by a temperature-sensitive oligonucleotide (according to the manufacturer). The polymerase becomes increasingly more active from a temperature of about 45 ° C, at a temperature of 65 ° C, no differences between polymerase Bst 2.0 and Bst 2.0 Warm start can be detected. To the extensive formation of by-products (eg, primer dimers) during the preparation phase of a reaction, Polymerase Bst 2.0 Warm start used. Deviations are specified.

Reaction was stopped by heating the reaction solution to above 80 ° C, for example 10 min at 95 ° C. At this temperature, polymerase Bst 2.0 irreversibly denatured and the result of the synthesis reaction can not be subsequently changed.

Reactions were carried out in a thermostat with a fluorescence measuring device. For this purpose, a commercial real-time PCR device was used, StepOne Plus (Applied Biosystems, Thermofischer). The reaction volume was 10 μl by default. Deviations are indicated.

Both endpoint determination and kinetic observations were made. For example, in endpoint determinations, the signal was registered by nucleic acids bound to nucleic acids, e.g. TMR (tetramethyl-rhodamine, also called TAMRA) or FAM (fluorescein). The wavelengths for excitation and measurement of the fluorescence signals of FAM and TMR are stored as factory settings in the StepOne Plus Real-Time PCR device. Also, an intercalating dye (EvaGreen) was used in end point measurements, e.g. when measuring a melting curve). EvaGreen is an intercalating dye and is an analog of the commonly used dye Sybrgreen, but with slightly less inhibition of polymerases. The wavelengths for excitation and measurement of the fluorescence signals from SybreGreen and EvaGreen are identical and are stored as factory settings in the StepOne Plus Real-Time PCR device. Fluorescence can be monitored continuously by means of built-in detectors, i. "Online" or "real-time" are detected. Since the polymerase synthesizes a double strand during its synthesis, this technique could be used for kinetic measurements (real-time monitoring) of the reaction. Due to some cross talk between color channels on the StepOne Plus device, partially increased basal signal intensity was observed in measurements where e.g. TMR-labeled primers in concentrations above 1 μmol / L (for example 10 μmol / L) were used. It has been observed that TMR signal in Sybre Green channel leads to increased baselines. These increased baseline values were taken into account in calculations.

The kinetic observations of response curves were routinely recorded by fluorescence signals from fluorescein (FAM-TAMRA Fret pair) and intercalating dyes (EvaGreen), respectively. Time-dependence of the signal course was registered (real-time signal acquisition with StepOne plus PCR device). An increase in the signal during a reaction compared to a control reaction was interpreted according to the structure of the approach. For example, an increase in the signal using Evagreen dye was interpreted as indicating increases in the amount of double-stranded nucleic acid chains during the reaction, and thus as a result of ongoing synthesis by DNA polymerase.

In some reactions, a melting curve determination was performed following the reaction. Such measurements allow conclusions about the presence of double strands, which can absorb, for example, intercalating dyes and thereby significantly enhance the signal intensity of dyes. As the temperature increases, the proportion of double strands decreases and the signal intensity also decreases. The signal is dependent on the length of nucleic acid chains and on the sequence composition. This technique is well known to a person skilled in the art.

For example, when using melting curve analysis in conjunction with reactions containing significant levels of modified nucleic acid chains (eg, controller oligonucleotides or primers), the EvaGreen dye signal was found to differ between B-form of DNA and A-form of DNA can behave modified nucleic acid chains. For example, in the B-form of the double-stranded nucleic acid chains (commonly believed for classical DNA segments), higher signal intensity has been observed than with double-stranded nucleic acid chains having the same sequence of nucleobases which may adopt an A-form-like conformation (eg, several '-O-Me modifications of nucleotides). This observation has been taken into account when using intercalating dyes.

If necessary, the reaction was analyzed by capillary electrophoresis and the length of fragments formed was compared to a standard. In preparation for capillary electrophoresis, the reaction mixture was diluted in a buffer (Tris-HCl, 20 mmol / l, pH 8.0, and EDTA, 20 mmol / l, pH 8.0) to such an extent that the concentration of labeled nucleic acids was ca. 20 nmol / l. Capillary electrophoresis was performed by GATC-Biotech (Konstanz, Germany) as a contracted service. According to the For example, capillary electrophoresis was performed on an ABI 3730 Cappilary Sequencer under standard conditions for Sanger sequencing using POP7 gel matrix, performed at about 50 ° C and constant voltage (about 10 kV). The conditions used led to denaturation of double strands, so that the single-stranded form of nucleic acid chains was separated in capillary electrophoresis. Electrophoresis is a standard technique in genetic analysis. Automated capillary electrophoresis is now routinely used in Sanger sequencing. The fluorescence signal is recorded continuously during capillary electrophoresis (usually using virtual filters) to produce an electrophorogram in which the signal intensity correlates with the duration of the electrophoresis. For shorter fragments, eg unused primers, an early signal peak is observed, with extended fragments there is a temporal shift of the signals proportional to the length of the extended region. Thanks to guided controls of known lengths, the length of extended fragments can be measured. This technique is known to a person skilled in the art and is also used by default in fragment length polymorphism.

Example 1 (Fig. 45)

Use of human genomic DNA as a source of target sequence

In this example, use of human genomic DNA (hgDNA) as the source of a target sequence is shown. The target sequence was a sequence segment of the Factor V Leiden gene (Homo sapiens coagulation factor V (F5), mRNA, referred to herein as the FVL gene).

Only the first amplification was performed. The example shows that a controller-dependent first amplification can be performed. In this example, no second amplification was performed.

• 5' GTAA GAGCAGATCC CTGGACAGGC AA GGAATACAGGTA - 3' (SEQ ID NO:1)

Target sequence:

• 5 'GTAA GAGCAGATCC CTGGACAGGC AA GGAATACAGGTA - 3' (SEQ ID NO: 1)

The binding sequence for the first primer oligonucleotide is underlined. The second primer oligonucleotide binds with its 3 'segment to the reverse complement of the double underlined sequence.

The first primer, the second primer, and controller oligonucleotide were designed and synthesized for FVL mutation variant of the gene.

• P1 F5-200-AE2053 5' AACTCAGACAAGATGTGATTTTTTTACCTGTAT [CUCU GAUGCUUC] 1TACCTGTATTCC 3'

The first primer oligonucleotide (SEQ ID NO: 2):

• P1 F5-200-AE2053 5 'AACTCAGACAAGATGTGATTTTTTTACCTGTAT [CUCU GAUGCUUC] 1 TACCTGTATTCC 3'

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The segment used as the primer in the reaction is underlined.

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

• 1 = C3-Linker diente zur Terminierung der Synthese des zweiten Primer-Verlängerungsproduktes.

This oligonucleotide comprises the following modifications:

• 1 = C3 Linker served to terminate the synthesis of the second primer extension product.

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)

Segment of the primer [CUCU GAUGCUUC] comprised 2'-O-Me modifications and served as a second primer region for binding the first region of the controller oligonucleotide:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)

Primer 2:
P2G3-5270-7063
5 'CTACAGAACTCAGACAAGATGTGAACTACAATGTT 6
GCTCATACTACAATGTCACTTACTGTAAGAGCAGA 3 '(SEQ ID NO: 3)

The segment used as the primer in the reaction is underlined.

• 6 = HEG-Linker
• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

This oligonucleotide comprises the following modifications:

• 6 = HEG linker
• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The following controller oligonucleotide (SEQ ID NO: 4) was used:
AD F5-1001-503
▫5 '[UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] AGGTAGAAGCATC AGAG X
3 '

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

The 5 'segment of the oligonucleotide [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] comprised 2'-O-Me nucleotide modifications:

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)
• X = 3'-Phosphat-Gruppe zur Blockade einer möglichen Verlängerung durch Polymerase.

modifications:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)
• X = 3'-phosphate group to block a possible extension by polymerase.

The nucleotides and nucleotide modifications are linked together with phosphodiester bonds. The 3 'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible extension by the polymerase.

The first primer comprises in its first region a sequence which can specifically bind to the sequence of the Factor V Leiden gene within the genomic DNA such that synthesis by a polymerase can be started. The second region of the first primer comprises a sequence which does not specifically hybridize to the sequence of FVL genes. Furthermore, the first primer comprises a further sequence segment which connects to the 5 'end of the second region. This segment does not participate in the specific amplification of the factor 5 Leiden segment. The function of this segment is mainly seen delaying side reactions.

The second primer comprises a segment in its 3 'segment which can specifically bind to the genomic DNA so that synthesis by a polymerase can be started. The 5 'segment of the second primer comprises a sequence that does not specifically hybridize to the sequence of FVL genes. During reverse synthesis, this sequence segment can be copied. The second primer comprises a further sequence segment which can hybridize specifically neither with the controller oligonucleotide nor with the first primer nor with the second primer. This segment was located at the 5 'end of the second primer and separated from the 5' end of the primer by a HEG linker. This segment is taking not participating in the specific amplification. The function of this segment is mainly seen in the delay of side reactions.

Controller oligonucleotide was designed such that a perfect match situation results in the sequence of Factor V Leiden mutation of the FVL gene. The controller oligonucleotide comprises first, second and third regions.

The genomic DNA used was WHO standard for FVL mutation. Prior to use in the reaction, DNA was denatured by heating (5 min at 95 ° C) and thus converted from the double-stranded state to single-stranded state. Attachment of this single-stranded hgDNA, a start-up nucleic acid chain was first created by a primer extension. Subsequently, an exponential amplification was carried out starting from this starting nucleic acid chain. The specificity of the amplification was demonstrated by melting curve analysis and Sanger sequencing with a sequencing primer.

All reactions were in amplification solution 1 carried out.

The dNTPs used included: dATP, dGTP, dCTP, dUTP (instead of dTTP).

As polymerase was Bst 2.0 Warm-start polymerase used by NEB.

The starting nucleic acid chain was generated as follows:

Approximately 50,000 haploid genome equivalents (HGE), 150 ng hgDNA, were placed in contact with the second primer (0.5 μmol / L) and Bst 2.0 Warm start polymerase (about 1 unit), and dNTPs (about 250 .mu.mol / l) under hybridization conditions (Amplifikationslösung 1 , Temperature of about 60 ° C) in 50 ul reaction volume and incubated for about 10 min. During this phase, the second primer is extended, using the genomic DNA as a template. The result is a primer extension product which can serve as a starting nucleic acid. After completion of this reaction, the reaction mixture was heated to 95 ° C for about 10 min to separate this starting nucleic acid chain template. This reaction mixture was frozen and used as a source of starting nucleic acid chain as needed.

The specific amplification of the target sequence of the FVL gene was carried out using 5 .mu.l of the reaction mixture with the starting nucleic acid chain (corresponds to about 5000 HGE).

The other reaction components (first primer, second primer, controller oligonucleotide, Eva-Green dye, Polymerase Bst.2.0 Warm Start, dNTPs) were added to give a final reaction volume of about 10 μl. The final concentrations of the components were: first primer: 5 μmol / l, second primer: 2 μmol / l, controller oligonucleotide: 1 μmol / l, Eva-Green dye (1:50), polymerase Bst.2.0 Warm Start (approx Units), dNTPs: approx. 250 μmol / l.

In the control approach no hgDNA was added.

The reaction was carried out in a Step-One Plus instrument (Thermofisher Scientific).

The reaction temperature was initially changed by cyclic changes (30 cycles) between 65 ° C (5 min, including detection step) and 55 ° C (1 min) and then held constant for 1 hr at 65 ° C (detection step every 2 min). The course of the reaction was monitored by signal detection of the EvaGreen dye. After completion of the reaction, the reaction mixture was first brought to 95 ° C for 10 minutes, and then a melting curve of the formed products was measured.

First, a preparation of a starting nucleic acid chain (schematically in 47 , Steps A to D shown). Subsequently, an exponential amplification was carried out with extension of the first primer and the second primer and the controller oligonucleotide.

As a result of the amplification, accumulation of amplification fragments occurs. The result of the detection of the amplification is in 45 to see. It can be seen that after about 2 hrs of the reaction a visible increase in fluorescence occurs (45 A). Subsequent melting curve analysis showed a specific melting curve with Tm of 84 ° C ( 45 B) ,

Sequence check ( 45C ):

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

Sequence checking was carried out by means of a sequencing primer:
SEQP1F5-35-X03
▫5'-AAG CTCGACAAAAGAAC TCAG AG TGTG CTCGAC AC TACCTGTATTCC ▫TTGCC 3 '(SEQ ID NO: 5)

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

For sequence testing, the reaction mixture (after measurement of the melting curve) was diluted with water (from about 1:10 to about 1: 100) and aliquots were mixed with a sequencing primer (added in concentration of 2 .mu.mol / l) mixed. This mixture was shipped by a commercial sequencing provider (GATC-Biotec) and sequenced by Sanger sequencing as order sequencing. The resulting electropherograms were checked for agreement with the FVL sequence gene. As a result of the reaction, sequence of the FVL gene was identified.

Example 2 (Figure 46):

Selective amplification reaction of sequence variants of a target sequence.

In this example, the influence of a sequence change in the template on the amplification was investigated. Upon extension of the first primer oligonucleotide, a complementary strand is formed which has a complementary sequence to the template and thus comprises these deviations in the sequence. In this way, it should be examined what effect such a mismatch between a resulting first primer extension product and a controller oligonucleotide has on the amplification. The position of the mismatch is in the 3 'direction of the first primer and is therefore not checked by the primer but by the controller oligonucleotide.

Discrimination between individual sequence variants of the target sequence thus takes place by means of a controller oligonucleotide using a uniform first and second primer.

Only the first amplification was performed. The example shows that a controller-dependent first amplification with discrimination between two target sequence variants can be performed. In this example, no second amplification was performed.

The following matrices were used:

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

Template (SEQ ID NO 6) with sequence composition leading to a first primer extension product with a perfect-match match with controller oligonucleotide:
M2SF5-M001-200
5 ' GCT CATA CTACAATGTCA CT TA CTGTAA GAGCAGA TCC CTGGACAGGC AA GGAATACAGGTA AAAAA 3' (SEQ ID NO: 6)

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

The binding sequence for the first primer oligonucleotide is underlined. The second primer oligonucleotide binds to the reverse complement of the double underlined sequence.

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

Template (SEQ ID NO 7) having sequence composition leading to a first primer extension product that mismatches with the controller oligonucleotide at a single base position (in bold):
M2SF5-WT01-200

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

The binding sequence for the first primer oligonucleotide is underlined. The second primer oligonucleotide binds to the reverse complement of the double underlined sequence.

The following primers were used:

The first primer oligonucleotide (SEQ ID NO: 2):
P1 F5-200-AE2053
5 'AACTCAGACAAGATGTGATTTTTTTACCTGTAT [CUCU GAUGCUUC] 1 TACCTGTATTCC 3'

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The segment used as the primer in the reaction is underlined.

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

• 1= C3-Linker diente zur Terminierung der Synthese des zweiten Primer-Verlängerungsproduktes.

This oligonucleotide comprises the following modifications:

• 1 = C3 linker served to terminate the synthesis of the second primer extension product.

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)

Segment of the primer [CUCU GAUGCUUC] comprised 2'-0-Me modifications and served as the second primer region for binding the first region of the controller oligonucleotide:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)

Primer 2:
P2G3-5270-7063
5 'CTACAGAACTCAGACAAGATGTGAACTACAATGTT 6 GCTCATACTACAATGTCACTTACTGTAAGAGCAGA 3' (SEQ ID NO: 8)

The segment used as the primer in the reaction is underlined.

• 6 = HEG-Linker
• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

This oligonucleotide comprises the following modifications:

• 6 = HEG linker
• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

AD-F5-1001-503 
 5' [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC]AGGTAGAAGCATC AGAG X
 3'

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The following controller oligonucleotide (SEQ ID NO: 4) was used:

 AD F5-1001-503 
 5 '[UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] AGGTAGAAGCATC AGAG X
 3 ' 

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

The 5 'segment of the oligonucleotide [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] comprised 2'-O-Me nucleotide modifications:

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)
• X = 3'-Phosphat-Gruppe zur Blockade einer möglichen Verlängerung durch Polymerase.

modifications:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)
• X = 3'-phosphate group to block a possible extension by polymerase.

The nucleotides and nucleotide modifications are linked together with phosphodiester bonds. The 3 'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible extension by the polymerase.

Four approaches have been prepared:

approach 1 contains as template nucleic acid sequence the template M2SF5-M001-200 (Perfect Match Situation) in a concentration of 300 fmol / l (corresponds to approximately 2x10 ^ 6 copies / batch).

approach 2 contains as template nucleic acid sequence the template M2SF5-M001-200 (Perfect Match Situation) in a concentration of 300 amol / l (corresponds to approx. 2x10 ^ 3 copies / batch).

approach 3 contains no matrix and thus forms a control.

approach 4 contains as starting nucleic acid chain the template M2SF5-WT01-200 (single mismatch situation) in 300 pmol / l concentration (about 2x 10 ^ 9 copies per batch).

primer 1 at 5 μmol / L, the controller oligonucleotide at 2 μmol / L and primer 2 used at 1 .mu.mol / l.

The other reaction conditions were: amplification solution 2 ,

To readjust the presence of genomic DNA in the assay, 100 ng of freshly denatured fish DNA (salmon DNA) was added per reaction.

The thermal reaction conditions were cyclic, alternating temperature changes, each followed by a 5 minute time interval at 65 ° C for a 2 minute time interval at 55 ° C. The amplification was followed for 100 cycles. Detection was carried out at 65 ° C for EvaGreen fluorescence signal.

The successful amplification could be detected by an increase in the EvaGreen fluorescence signal over time.

Temperature changes and real-time monitoring were performed with the StepPne Plus Real-Time PCR device from Thermofisher.

46A shows a typical course of the EvaGreen signal. On the Y -Axis is the increase of the fluorescence signal (delta Rn) and on the X -Axis is the reaction time (as cycle number) plotted. Arrows mark individual reaction approaches. The selected positions belong to the following approaches: Arrow 1: 300 fmol / l perfect match template (about 2x10 ^ 6 copies / batch) Arrow 2: 300 amol / l perfect match die (about 2x10 ^ 3 copies / batch) Arrow 3: no die Arrow 4: 300 pmol / l mismatch template (about 2x 10 ^ 9 copies per batch).

The increase of the fluorescence signal is seen in both the perfect match and the mismatch variant of the template, with the signal of the mismatch variant (FIG. 4 ) despite 100 times over appears later. It can be seen that the mismatch amplification signals are significantly delayed from the perfect match amplification signal. With the single mismatch, a delay of about 15 cycles is observed. The time delay (= number of cycles) is a direct measure of the discrimination in the amplification. Further quantification of discrimination in amplification can be achieved by comparison with a template concentration series under perfect match amplification.

Using a Perfect-Match template results in the synthesis of a complementary strand of a primer extension product. This extension product is complementary to both the Perfect Match template and the controller oligonucleotide used.

In contrast, using a mismatch sequence, the synthesis of the first primer extension product results in the generation of a complementary strand of the extension product which, while having complete complementarity to the mismatch template, is thereby complemented by the third region of the controller -Oligonucleotide differs. This departure occurs in the 5 'segment of the extension product, which should react with controller oligonucleotide to allow strand displacement to proceed. As shown in the present example, the mismatch interferes with strand displacement by the controller oligonucleotide.

The control reactions with a Perfect Match matrix (arrows 1 and 2 ) showed a concentration dependence of the amplification. With decreasing concentration, reaction took longer time to synthesize a sufficient amount of the nucleic acid to be amplified to raise the signal above baseline levels.

This result illustrates the importance of the base composition in the controller oligonucleotide: deviations from the complementarity between the controller oligonucleotide and the primer extension product can lead to slowing or even interrupting the amplification.

In this example, it was shown that although the sequence ends of the perfect match template and the mismatch template were completely identical and thus the potential for binding of both primer oligonucleotides was the same, both reactions proceeded completely differently: with a complete complementarity between the Controller oligonucleotide and the 5 'segment of the extension product of the first primer oligonucleotide proceeded according to the amplification. An interruption of strand displacement by a sequence deviation (in this case by a mismatch) led to the suppression of the amplification.

Example 3 (Figures 47-51):

Use of two amplifications of a first amplification and followed by a second amplification

In this embodiment it is demonstrated that amplification products of the first amplification (first amplification) can be used by a downstream, classical PCR (second amplification) as starting nucleic acid chains. Thus, a total of two separate amplification reactions were carried out: first a first amplification, then a second amplification.

First amplification

First, a first amplification was performed using a single-stranded nucleic acid chain (here as a single-stranded starting nucleic acid chain 1.1 ) comprising a target sequence using a first amplification system. The reaction conditions used (temperature of 55 ° C and 65 ° C) do not permit spontaneous separation of the double strand formed during the reaction, comprising the first primer extension product and the second primer extension product, in the absence of a controller oligonucleotide.

• • Ein erstes Primer-Oligonukleotid,
• • ein zweites Primer-Oligonukleotid,
• • ein Controller-Oligonukleotid und
• • eine Polymerase, (Bst 2.0 Warm Start Polymerase), welche Strangverdrängungseigenschaften hat
• • sowie notwendige Substrate für Polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) und geeignete Puffer-Systeme (Die Reaktion wurde im Isothermal-Puffer 1x (NEB) durchgeführt).

The first amplification system included the following components:

• A first primer oligonucleotide,
• A second primer oligonucleotide,
• A controller oligonucleotide and
• • a polymerase, (Bst 2.0 Warm start polymerase), which has strand displacement properties
• • as well as necessary substrates for polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) and suitable buffer systems (The reaction was carried out in isothermal buffer 1x (NEB) performed).

The progress of the reaction was detected by means of EvaGreen at 65 ° C.

At the concentration ratios of the first primer oligonucleotide (5 μmol / l) and the controller oligonucleotide (2 μmol / l), the first primer is in relative excess to the controller oligonucleotide. The melting temperature of the complex comprising the first primer oligonucleotide and the controller oligonucleotide was about 63 ° C (Tm), measured in the same reaction solution.

In the first amplification reaction two temperature ranges were used:

55 ° C (2 min) and 65 ° C (2 min). At temperature step 55 ° C, the controller oligonucleotide was completely in complex with the first primer oligonucleotide. This did not allow the controller oligonucleotide to bind to the first primer extension product. At the second temperature step (65 ° C), this complex was able to dissociate at least partially from the complex, so that free, single-stranded controller oligonucleotides were available in preparation. Such a free controller oligonucleotide may either form a complex with a new first primer oligonucleotide or anneal to the first primer extension product. Contact with the first primer extension product begins by complementary binding of the sequence segments of the first region of the controller oligonucleotide to the corresponding sequence segments of the second primer region, which is not copied by polymerase.

The individual synthesis processes and strand separation processes essentially comprise the following processes:

The first primer oligonucleotide can be attached to the 3 'segment of the nucleic acid chain provided (starting nucleic acid chain 1.1 ) bind specifically and are extended by polymerase (this reaction proceeds predominantly at 55 ° C). The synthesis proceeds to the end of the template strand (5 'region of the starting nucleic acid chain). This resulted in a first Priemr extension product. The controller oligonucleotide can bind to this first primer extension product by displacing the template strand (here initially the starting nucleic acid chain) by complementary base pairing (this reaction proceeds predominantly at 65 ° C). The separation of the 3 'segment of the first primer extension product (which was not bound by the controller) from the complex with the template strand was predominantly temperature-induced at 65 ° C. (the Tm of this 3 'segment was below 65 ° C). The 3 'segment of the first primer extension product thereby became single-stranded. The first primer extension product was thus in complex with the controller oligonucleotides.

The second primer oligonucleotide was now able to complementarily bind to this 3 'segment of the synthesized first primer extension product and to initiate the synthesis of the second primer extension product by the polymerase (this step can be performed at both 55 ° C be done at 65 ° C). The synthesis was performed while displacing the controller oligonucleotide from binding to the first primer extension product. The polymerase used in the first primer extension reaction (Bst 2.0 Warm Start) had a strand displacement characteristic. The synthesis of the second primer extension product was carried out up to and including the first region of the first primer, which hereby was copied from polymerase. The synthesis of the second primer extension product was performed by C3 Linker in the first. Primer extension product so that the second portion of the first primer extension product was not copied by polymrease. The result was the second primer extension product, which formed a double strand with the first primer extension product (Tm of this double strand = first amplification product was about 79 ° C). Repeated heating of the reaction mixture to 65 ° C re-bound the controller oligonucleotide to the first primer extension product so that now the second primer extension product was released from its binding to the first primer extension product.

As a result of the release of the second primer extension product, this product could now serve as a template: the second primer extension product comprises in its 3 'segment a sequence which is complementary to the first region of the first primer and thus capable of forming a new primer To bind oligonucleotide and start the synthesis by means of polymerase.

As a result, repeated synthesis and strand separation events occurred, with both synthesized primer extension products occurring as templates in subsequent cycles.

The first amplification product could be prepared starting from the starting nucleic acid chain ( 1.1 ) were synthesized using primers provided with the aid of the provided controller oligonucleotide until the desired amount of copies were obtained (here, the concentration of the first amplification product 1.1 was at the end of the first amplification about 0.8 - 1 .mu.mol / l. which corresponds to approximately 1000,000,000,000 copies in 10 μl of reaction mixture). The number of copies was estimated by way of illustration from concentration.

The reaction was stopped by heating to 95 ° C for 10 min, denaturing the first polymerase. Melting-fast analysis was then performed to confirm the specific amplification. The reaction mixture was frozen and used as needed in the second amplification.

Second Amplification (PCR)

After completion of the first amplification, a portion of the first reaction mixture was added to the second amplification. The first amplification fragment synthesized during the first amplification ( 1.1 ) as a second starting nucleic acid chain ( 2.1 ) for the second amplification.

Several dilution steps of the first completed amplification run were added to the second amplification reaction, resulting in a dilution series.

It should be noted that no purification of formed first amplification fragments ( 1.1 ), but only a dilution. So that all unused components of the first amplification system - albeit in diluted form - were also present in the second amplification step.

The PCR reaction conditions used (three temperature ranges of 55 ° C (1 min) and 72 ° C (3 min) and 95 ° C (20 sec) allowed standard PCR (second amplification) to be performed.

• • Ein drittes Primer-Oligonukleotid,
• • ein viertes Primer-Oligonukleotid,
• • eine Polymerase, (Taq Polymerase), welche eine thermostabile Polymerase ist
• • sowie notwendige Substrate für Polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) und geeignete Puffer-Systeme.

The second amplification system each comprised the following components:

• A third primer oligonucleotide,
• A fourth primer oligonucleotide,
• A polymerase, (Taq polymerase), which is a thermostable polymerase
• • as well as necessary substrates for polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) and suitable buffer systems.

Typical PCR concentrations of individual components were used: PCR primer in final concentrations of 0.5 .mu.mol / l, and the Taq polymerase in concentration (1,100 dilution, equivalent to about 1 unit / reaction) and dNTPs (A, G, C, T) in concentration of about 200μmol / l. The reaction was in isothermal buffer 1x (NEB).

The second amplification was carried out using this first amplification fragment as starting nucleic acid chain ( 2.1 ) and using a second amplification system. The progress of the synthesis was detected by means of EvaGreen at 72 ° C. As a control reaction were NTC and start nucleic acid chain 1.1 used, which can also be used as a template in the second amplification system.

The number of cycles required in the second amplification, up to the rise of the specific signal, was measured and compared to reference values. From the comparison of the number of cycles required for the detectable amplification, the successful use of the first amplification fragment ( 1.1 ) closed during the PCR.

In detail, the following was done:

The first amplification product of a controller oligonucleotide-based first amplification reaction was diluted and amplified as a template (input) under typical PCR conditions. The detection in the context of the downstream PCR is carried out in this example with the intercalating dye EvaGreen. By comparison with a number of different control approaches, we show that a signal only arises in the downstream PCR if the controller oligonucleotide-based amplification reaction was successful. For this reason, the downstream, classical PCR can also be called a detection PCR.

The amplification product of the controller oligonucleotide-based amplification reaction was essentially prepared as described in Example 2.

The following primer oligonucleotides were used:

The first primer oligonucleotide:
P1F5-200-AE205
CACTTAC GACA22CAAGA TTTTTT TACCTGTAT [CUCU GAUGCUUC] 1 TACCTGTATTCC

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The segment used as the primer in the reaction is underlined.

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

• 1= C3-Linker diente zur Terminierung der Synthese des zweiten Primer-Verlängerungsproduktes.
• 2 = 2'-deoxy-lnosine

This oligonucleotide comprises the following modifications:

• 1 = C3 linker served to terminate the synthesis of the second primer extension product.
• 2 = 2'-deoxy-inosine

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)

Segment of the primer [CUCU GAUGCUUC] comprised 2'-O-Me modifications and served as the second primer region for binding the first region of the controller oligonucleotide:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)

The second primer oligonucleotide:
P2G3-5270-7063
5'CTACAGAACTCAGACAAGATGTGAACTACAATGTT6 GCTCATACTACAATGTCACTTACTGTAAGAGCAGA 3 '(SEQ ID NO: 8)

The segment used as the primer in the reaction is underlined.

• 6 = HEG-Linker
• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

This oligonucleotide comprises the following modifications:

• 6 = HEG linker
• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

 AD-F5-1001-503
 5' [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC]AGGTAGAAGCATC AGAG X
 3'

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The following controller oligonucleotide (SEQ ID NO: 4) was used:

 AD F5-1001-503
 5 '[UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] AGGTAGAAGCATC AGAG X
 3 ' 

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

The 5 'segment of the oligonucleotide [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] comprised 2'-O-Me nucleotide modifications:

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)
• X = 3'-Phosphat-Gruppe zur Blockade einer möglichen Verlängerung durch Polymerase.

modifications:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)
• X = 3'-phosphate group to block a possible extension by polymerase.

The nucleotides and nucleotide modifications are linked together with phosphodiester bonds. The 3 'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible extension by the polymerase.

M2SF5-M001-200

 5' GCT CATA CTACAATGTCA CT TA CTGTAA GAGCAGATCC CTGGACAGGC AA
 GGAATACAGGTA AAAAA 3'

Start nucleic acid chain ( 1.1 ) with pefekt-match match to the controller.

 M2SF5-M001-200

 5 'GCT CATA CTACAATGTCA CT TA CTGTAA GAGCAGATCC CTGGACAGGC AA
 GGAATACAGGTA AAAAA 3 ' 

The starting nucleic acid chain was used at a concentration of about 1 pM.

 5' GCT CATA CTACAATGTCA CT TA CTGTAA GAGCAGATCC CTGGACAGGC AA
 GGAATACAGGTA 3'

(die nicht kopierbaren Sequenzanteile des zweiten Primer sind hier nicht dargestellt).As a result, an amplification fragment is expected to give the second primer extension product with the sequence:

 5 'GCT CATA CTACAATGTCA CT TA CTGTAA GAGCAGATCC CTGGACAGGC AA
 GGAATACAGGTA 3 ' 

(the non-duplicable sequence portions of the second primer are not shown here).

The PCR was performed with 3 different primer pairs. The primer pairs capture different sequence regions of the amplification product. For a better understanding, the primer binding sites on the template strand (= amplification product) are also labeled for each primer pair.

For the PCR were used:

PCR primer pair 1 consisting of the third primer oligonucleotide SeqP1F5 300-35x (SEQ ID NO XY) and the fourth primer oligonucleotide P2-4401-601 TMR (SEQ ID NO XY):

SeqP1F5 300-35x

 5' ACGAAGCTCGCAAGAACTCAGAGTGTGCTCGACACTACCTGTATTCC 3'

The third primer oligonucleotide (SEQ ID NO XY)

 SeqP1F5 300-35x

 5 'ACGAAGCTCGCAAGAACTCAGAGTGTGCTCGACACTACCTGTATTCC 3' 

 P2-4401-601 TMR
 5' GCTCATACTACAATGTCACTTAC 3'

The fourth primer oligonucleotide (SEQ ID NO XY)

 P2-4401-601 TMR
 5 'GCTCATACTACAATGTCACTTAC 3' 

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)
• TMR = Tetramethylrhodamine am 5'-Ende des Stranges angehängt (P2-4401-601 TMR)

Each with:

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)
• TMR = tetramethylrhodamine attached at the 5 'end of the strand (P2-4401-601 TMR)

These two primers bind to the template strand in the following manner (shown here as the second primer extension product of the first amplification):
5 ' GCT CATA CTACAATGTCA CT TA C TGTAA GAGCAGATCC CTGGACAGGC AA GGAATACAGGTA 3'

In the control approach, primers of this primer pair can also be attached to the starting nucleic acid chain 1.1 bind as follows:
M2SF5-M001-200
5 ' GCT CATA CTACAATGTCA CT TA C TGTAA GAGCAGATCC CTGGACAGGC AA GGAATACAGGTA AAAAA 3'

The binding sequence for the first primer oligonucleotide is underlined. The second primer oligonucleotide binds to the reverse complement of the double underlined sequence.

PCR primer pair 2 consisting of the third primer oligonucleotide P3F5D-600-203 (SEQ ID NO XY) and the fourth primer oligonucleotide P2-4401-601 TMR (SEQ ID NO XY):

 P3F5D-600-203
 TMR - 5'
 GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAATGTCCAGGGA 3'

The third primer oligonucleotide (SEQ ID NO XY)

 P3F5D-600-203
 TMR - 5 '
 GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAATGTCCAGGGA 3 ' 

 P2-4401-601 TMR
 TMR - 5' GCTCATACTACAATGTCACTTAC 3'

The fourth primer oligonucleotide (SEQ ID NO XY)

 P2-4401-601 TMR
  TMR - 5 'GCTCATACTACAATGTCACTTAC 3' 

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

Each with:

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

• TMR = Tetramethylrhodamine am 5'-Ende des Stranges angehängt (P3F5D-600-203) und des P2-4401-601 TMR

modifications:

• TMR = tetramethylrhodamine attached at the 5 'end of the strand (P3F5D-600-203) and the P2-4401-601 TMR

These two primers bind to the template strand in the following manner (shown here as the second primer extension product of the first amplification):
5 ' GCT CATA CTACAATGTCA C T TA CTGTAA GAGCAGA TCC CTGGACA GGC AA GGAATACAGGTA 3'

In the control approach, primers of this primer pair can also be attached to the starting nucleic acid chain 1.1 bind as follows:

The binding sequence for the first primer oligonucleotide is underlined.

The second primer oligonucleotide binds to the reverse complement of the double underlined sequence.

PCR primer pair 3 consisting of the third primer oligonucleotide SeqP1F5-35-X02 (SEQ ID NO XY) and the fourth primer oligonucleotide P2-4401-601 TMR (SEQ ID NO XY):

 SeqP1F5-35-X02
 5' AAGCTCGACAAAAGAACTCAGAGTGTGCTCGACACTACCTGTATTCCTTG 3'

The third primer oligonucleotide (SEQ ID NO XY)

 SeqP1F5-35-X02
 5 'AAGCTCGACAAAAGAACTCAGAGTGTGCTCGACACTACCTGTATTCCTTG 3' 

 P2-4401-601 TMR
 TMR - 5' GCTCATACTACAATGTCACTTAC 3'

The fourth primer oligonucleotide (SEQ ID NO XY)

 P2-4401-601 TMR
 TMR - 5 'GCTCATACTACAATGTCACTTAC 3' 

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)
• TMR = Tetramethylrhodamine am 5'-Ende des Stranges angehängt (P2-4401-601 TMR)

Each with:

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)
• TMR = tetramethylrhodamine attached at the 5 'end of the strand (P2-4401-601 TMR)

These two primers bind to the template strand in the following manner (shown here as the second primer extension product of the first amplification):

In the control approach, primers of this primer pair can also be attached to the starting nucleic acid chain 1.1 bind as follows:
M2SF5-M001-200

The binding sequence for the first primer oligonucleotide is underlined.

The second primer oligonucleotide binds to the reverse complement of the double underlined sequence.

The nucleotides and nucleotide modifications are linked to each other with phosphodiester bonds.

Taq polymerase (NEB, catalog # M0273S) was used in the PCR. Polymerase dilutions of 1: 100 were used per reaction.

The amplification product from a controller oligonucleotide-based amplification reaction (first amplification) was used in the PCR in dilutions of 1: 5,000 to 1: 500,000,000.

As a control, also reaction mixtures were investigated which contained no template for amplification (NTC = no template control = negative control).

In addition, it was tested in further control approaches, whether the controller oligonucleotide can serve as a template for PCR primer. For this purpose, the controller oligonucleotide (SEQ ID NO XY = here as "controller 503 Previously used during the controller oligonucleotide-based amplification, in concentrations of 1 .mu.mol / l to 1 pmol / l offered for amplification in the PCR.

In a further control, defined amounts of the template M2SF5-M001-200 were offered for amplification in the PCR (positive control). This control demonstrated the performance of the PCR primer pairs (under ideal conditions). In addition, one could also quantitate a sample after a first amplification by comparing the obtained Ct values with a standard dilution series of the positive control. Concretely, the template M2SF5-M001-200 was tested in concentrations of 1 nmol / l to 1 fmol / l.

The third and the fourth primer oligonucleotides were used in concentrations of 0.5 .mu.mol / l.

• • 1x Isothermal Puffer (New England Biolabs, catalog # B0537S; in einfacher Konzentration enthält der Puffer:
  • 20 mM Tris-HCI
  • 10 mM (NH4)2SO4
  • 50 mM KCl
  • 2 mM MgSO4
  • 0.1% Tween® 20
  • pH 8.8@25°C)
• • dNTP (dATP, dCTP, dTTP, dGTP), je 200 µmol/l
• • EvaGreen Farbstoff (Farbstoff wurde entsprechend Anleitung des Herstellers in Verdünnung 1:50 eingesetzt)

The further reaction conditions were:

• • 1x isothermal buffer (New England Biolabs, catalog # B0537S; in simple concentration the buffer contains:
  • 20 mM Tris-HCl
  • 10mM (NH4) 2SO4
  • 50 mM KCl
  • 2mM MgSO4
  • 0.1% Tween® 20
  • pH 8.8 @ 25 ° C)
• • dNTP (dATP, dCTP, dTTP, dGTP), 200 μmol / l each
• • EvaGreen dye (dye was used according to manufacturer's instructions in dilution 1:50)

• • 20 Sekunden 95 °C zur Aktivierung des Systems
• • 30 Zyklen mit jeweils folgendem Temperatur-Zeit-Profil
  • ◯ 1 min 55 °C
  • ◯ 3 min 72°C
  • ◯ 20 Sekunden 95°C
• • 10 min 72 °C zur Vereinheitlichung der Amplifikationsprodukte
• • Schmelzkurvenanalyse

The thermal reaction conditions were chosen as follows:

• • 20 seconds 95 ° C to activate the system
• • 30 cycles, each with the following temperature-time profile
  • ◯ 1 min 55 ° C
  • ◯ 3 min 72 ° C
  • ◯ 20 seconds 95 ° C
• • 10 min 72 ° C to standardize the amplification products
• • melting curve analysis

The amplification was typically over 30 cycles, i. 30 x (1 min 55 ° C + 3 min 72 ° C + 20 s 95 ° C) = approx. 30 x 260 s = 130 min followed. The successful amplification could be detected by an increase in the EvaGreen fluorescence signal over time.

Analysis of detection PCR

• • Fluoreszenzsignal eines interkalierenden Farbstoffes (EvaGreen)
• • Schmelzkurvenanalyse von den entstandenen Amplifikationsprodukten

The following techniques were used to analyze the detection PCR and to evaluate the resulting amplification products:

• Fluorescence signal of an intercalating dye (EvaGreen)
• Melting curve analysis of the resulting amplification products

In the analysis of PCR, we only discuss data obtained with a 1: 100 dilution of Taq polymerase.

In the control experiments with template (M2SF5-M001-200), it was possible to verify that all three PCR primer pairs selected could generate a specific PCR product under PCR conditions used. It was also possible to verify that all three primer pairs were unable to form a product based on controller oligonucleotide. This was an expected result since all used "third" primers bind in the modified portion of the controller oligonucleotide.

47A shows a typical time course of the EvaGreen fluorescence signal used in the amplification of diluted controller oligonucleotide amplification approaches, by primer pair 1 was obtained. On the Y -Axis is the fluorescence signal of the EvaGreen-dye and on the X -Axis is the reaction time (as cycle number) plotted. Arrows mark different amplification approaches. The arrows include the following approaches: position Dilution of the Controller Oligonucleotide Amplification Approach in the Detection PCR Approach Arrow 1 about 1: 5,000 Arrow 2 about 1: 50,000 Arrow 3 about 1: 500,000 Arrow 4 about 1: 5,000,000 Arrow 5 about 1: 50,000,000 Arrow 6 0 mol / l = negative control

Arrow 6 marks a negative control approach that does not contain a template and therefore does not generate an amplification signal during the course of the experiment.

Depending on the chosen dilution of the controller oligonucleotide amplification approach, time-shifted amplification signals are observed. For PCR primer pair 1 Under the chosen conditions, dilutions of 1: 5,000 to 1: 50,000,000 can be well resolved. A dilution of 1: 50,000,000 produces an amplification signal that is marginally different from the negative control (see arrow 5 ).

47 B shows the associated melting curve analysis for the reactions 1 to 5 and makes it clear that specific amplification products were formed (Tm = 82.7 ° C).

47 B shows the melting curves of the amplification products of primer pair 1 out 47 A , On the Y -Axis is the derivative of the fluorescence signal against the temperature and on the X -Axis is the temperature applied. A uniform peak was found, marked with arrow 1 , The peak at position 1 with a melting point near 82.7 ° C belongs to the amplification product of the primer pair 1 , Melting curve analysis indicates specific amplification.

48A shows a typical time course of the EvaGreen fluorescence signal used in the amplification of diluted controller oligonucleotide amplification approaches, by PCR primer pair 2 was obtained. On the Y -Axis is the fluorescence signal of the EvaGreen-dye and on the X -Axis is the reaction time (as cycle number) plotted. Arrows mark different amplification approaches. The arrows include the following approaches: position Dilution of the Controller Oligonucleotide Amplification Approach in the Detection PCR Approach Arrow 1 about 1: 5,000 Arrow 2 about 1: 50,000 Arrow 3 about 1: 500,000 Arrow 4 about 1: 5,000,000 Arrow 5 0 mol / l = negative control

arrow 5 marks a negative control approach that does not contain a template and therefore does not generate an amplification signal during the course of the experiment.

Depending on the chosen dilution of the controller oligonucleotide amplification approach, time-shifted amplification signals are observed. For PCR primer pair 2 Under the chosen conditions, dilutions of 1: 5,000 to 1: 5,000,000 can be well resolved. A dilution of 1: 5,000,000 produces an amplification signal that just stands out from the negative control arrow 4 ).

48 B shows the associated melting curve analysis for the reactions 1 to 4 and makes it clear that specific amplification products have emerged.

48 B shows the melting curves of the amplification products of PCR primer pair 2 out 48 A , On the Y-axis the derivative of the fluorescence signal is against the temperature and on the X-axis the temperature is plotted. A uniform peak was found, marked with arrow 1 , The peak at position 1 with a melting point near 81.9 ° C belongs to the amplification product of the PCR primer pair 2 , Melting curve analysis indicates specific amplification.

49A shows a typical time course of the EvaGreen fluorescence signal used in the amplification of diluted controller oligonucleotide amplification approaches, by PCR primer pair 3 was obtained. On the Y -Axis is the fluorescence signal of the EvaGreen-dye and on the X -Axis is the reaction time (as cycle number) plotted. Arrows mark different amplification approaches. The arrows include the following approaches: position Dilution of the Controller Oligonucleotide Amplification Approach in the Detection PCR Approach Arrow 1 about 1: 5,000 Arrow 2 about 1: 50,000 Arrow 3 about 1: 500,000 Arrow 4 about 1: 5,000,000 Arrow 5 about 1: 50,000,000 Arrow 6 about 1: 500,000,000 Arrow 7 0 mol / l = negative control

arrow 7 marks a negative control approach that does not contain a template and therefore does not generate an amplification signal during the course of the experiment.

Depending on the chosen dilution of the controller oligonucleotide amplification approach, time-shifted amplification signals are observed. For primer pair 3 Under the chosen conditions, dilutions of 1: 5,000 to 1: 500,000,000 can be well resolved. A dilution of 1: 500,000,000 produces an amplification signal that just stands out from the negative control (see arrow 6 and 7 ).

49 B shows the associated melting curve analysis for the reactions 1 to 6 and makes it clear that specific amplification products have emerged.

49B shows the melting curves of the amplification products of PCR primer pair 3 out 49 A , On the Y -Axis is the derivative of the fluorescence signal against the temperature and on the X -Axis is the temperature applied. A uniform peak was found, marked with arrow 1 , The peak at position 1 with a melting point near 81.2 ° C belongs to the amplification product of the PCR primer pair 3 , Melting curve analysis indicates specific amplification.

Analysis of the fluorescence signals over the course of the amplification (amplification plots) and the melting curve analysis at the end of the amplification showed that the PCR primer pairs 1 to 3 each were capable of using diluted amplification products resulting from the first amplification reaction as template.

Depending on the dilution chosen, time-delayed amplification signals are produced which are typical for a dilution series. By the PCR conditions selected here 5,000,000-fold diluted amplification products can be detected. The primer pair 3 has the highest sensitivity and can even detect amplification products at 500,000,000-fold dilution.

50 shows as control experiments with template (M2SF5- M001 -200), which in 1 nmol / l (arrow 1 ) and 10 pmol / l (arrow 2 ) and NTC controls (arrow 3 ).

50 A provides results for PCR primer pair 1 represents.

50 B provides results for PCR primer pair 2 represents.

50 C provides results for PCR primer pair 3 represents.

From the comparison of the time of rise of the fluorescence signal between control experiments (template) and the performed second amplification from first amplification fragments, it can be seen that the first amplification caused an amplification of amplification fragments which used as template in the second amplification could become.

51 shows course of the first amplification (before PCR).

One recognizes a signal rise, which was interpreted as an enrichment of the copy number in the first amplification. arrow 1 corresponded to a start nucleic acid chain approach 1.1 , Arrow 2 = NTC control.

Example 4 (Figures 52-56):

Combination of the first amplification and the second amplification in the same approach (homogeneous assay)

In this embodiment, we demonstrate how the controller oligonucleotide-based amplification reaction can be combined with downstream, classical PCR in a homogeneous assay format. Here, we use the highly selective controller oligonucleotide-based amplification reaction in the early amplification phase and then switch to a classical PCR for amplification and detection of the amplification products. In contrast to embodiment X (= heterogeneous assay format), the combination in this embodiment takes place in a homogeneous assay format. All assay components are already available at the start of the reaction, switching between controller-oligonucleotide-based amplification and classical PCR is achieved by a change in the temperature regime during the course of the reaction.

First became the first amplification product 1.1 starting from a single-stranded starting nucleic acid chain ( 1.1 ) amplified controller oligonucleotide-based amplification reaction for 15 to 30 cycles followed by 30 classical PCR cycles to further amplify and detect the pre-amplified product. For the detection we use the intercalating dye EvaGreen in this example.

• • Ein erstes Primer-Oligonukleotid,
• • ein zweites Primer-Oligonukleotid,
• • ein Controller-Oligonukleotid und
• • eine Polymerase, (Bst 2.0 Warm Start Polymerase), welche Strangverdrängungseigenschaften hat
• • sowie notwendige Substrate für Polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) und geeignete Puffer-Systeme (Die Reaktion wurde im Isothermal-Puffer 1x (NEB) durchgeführt).

The first amplification system included the following components:

• A first primer oligonucleotide,
• A second primer oligonucleotide,
• A controller oligonucleotide and
• • a polymerase, (Bst 2.0 Warm start polymerase), which has strand displacement properties
• • as well as necessary substrates for polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) and suitable buffer systems (The reaction was carried out in isothermal buffer 1x (NEB) performed).

• • Ein drittes Primer-Oligonukleotid,
• • ein viertes Primer-Oligonukleotid
• • eine Polymerase, (Taq Polymerase), welche eine thermostabile Polymerase ist
• • sowie notwendige Substrate für Polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) und geeignete Puffer-Systeme.

The second amplification system each comprised the following components:

• A third primer oligonucleotide,
• A fourth primer oligonucleotide
• A polymerase, (Taq polymerase), which is a thermostable polymerase
• • as well as necessary substrates for polymerase (dNTP's: dATP, dCTP, dGTP, dTTP) and suitable buffer systems.

Both amplification systems were together before the start of the first amplification.

In this example, the second primer oligonucleotide was used for both amplifications. The fourth primer oligonucleotide is thus identical to the second primer oligonucleotide.

The third primer oligonucleotide was varied.

In this example, we present 4 different primers, each individually used per approach as "the third primer oligonucleotide".

The respective third primer oligonucleotide differs from the first primer oligonucleotide. Thus, each reaction approach involves three primers: a first primer oligonucleotide and a second primer oligonucleotide (as components of the first amplification system) and the third primer oligonucleotide used as a specific primer of the second amplification system. The role of the fourth primer was taken over by the second primer of the first amplification system, which was also suitable for the downstream PCR reaction. For a better understanding, the primer binding sites on the template strand (= amplification product) are labeled for each primer combination.

Following start nucleic acid chain ( 1.1 ) was used as a single-stranded template:

Template (SEQ ID NO XY) having sequence composition leading to a first primer extension product having a match-perfect match with the controller oligonucleotide:
M2SF5-M001-200

The binding sequence for the first primer oligonucleotide is underlined. The second primer oligonucleotide binds to the reverse complement of the double underlined sequence. The binding sequence for the third primer oligonucleotide (P3F5D-600-203) is shown in bold italics.

The first amplification system:

The first primer oligonucleotide:
P1F5-200-AE205
▫CACTTAC GACA22CAAGA TTTTTT TACCTGTAT [CUCU GAUGCUUC] 1 TACCTGTATTCC

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The segment used as the primer in the reaction is underlined.

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

1. 1 = C3-Linker diente zur Terminierung der Synthese des zweiten Primer-Verlängerungsproduktes.
2. 2 = 2'-deoxy-Inosine

This oligonucleotide comprises the following modifications:

1. 1 = C3 linker served to terminate the synthesis of the second primer extension product.
2. 2 = 2'-deoxy-inosine

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)

Segment of the primer [CUCU GAUGCUUC] comprised 2'-O-Me modifications and served as the second primer region for binding the first region of the controller oligonucleotide:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)

The second primer oligonucleotide:
P2G3-5270-7063
5 'CTACAGAACTCAGACAAGATGTGAACTACAATGTT 6
GCTCATACTACAATGTCACTTACTGTAAGAGCAGA 3 '(SEQ ID NO: 8)

The segment used as the primer in the reaction is underlined.

• 6 = HEG-Linker
• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

This oligonucleotide comprises the following modifications:

• 6 = HEG linker
• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

 AD-F5-1001-503
 5' [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC]AGGTAGAAGCATC AGAG X
 3'

• A = 2'-deoxy-Adenosin
• C = 2'-deoxy-Cytosin
• G = 2'-deoxy-Guanosin
• T = 2'-deoxy-Thymidin (Thymidin)

The following controller oligonucleotide (SEQ ID NO: 4) was used:

 AD F5-1001-503
 5 '[UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] AGGTAGAAGCATC AGAG X
  3 ' 

• A = 2'-deoxy-adenosine
• C = 2'-deoxy-cytosine
• G = 2'-deoxy-guanosine
• T = 2'-deoxy-thymidine (thymidine)

The 5 'segment of the oligonucleotide [UAAUCUGUAA GAGCAGAUCC CUGGACAGGC AA GGAAUAC] comprised 2'-O-Me nucleotide modifications:

• A = (2'-O-Methyl-Adenosine)
• G = (2'-O-Methyl-Guanosine)
• C = (2'-O-Methyl-Cytosine)
• U = (2'-O-Methyl-Uridine)
• X = 3'-Phosphat-Gruppe zur Blockade einer möglichen Verlängerung durch Polymerase.

modifications:

• A = (2'-O-methyl-adenosine)
• G = (2'-O-methyl-guanosine)
• C = (2'-O-methyl-cytosine)
• U = (2'-O-methyl uridine)
• X = 3'-phosphate group to block a possible extension by polymerase.

The nucleotides and nucleotide modifications are linked together with phosphodiester bonds. The 3 'end of the controller oligonucleotide is blocked with a phosphate group to prevent possible extension by the polymerase.

• • Das dritte Primer-Oligonukleotid (SEQ ID NO XX):
  • P3F5D-600-201 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA CTGTCCAGGGATC 3'
  • A = 2'-deoxy-Adenosin
  • C = 2'-deoxy-Cytosin
  • G = 2'-deoxy-Guanosin
  • T = 2'-deoxy-Thymidin (Thymidin)

The second amplification system specifically comprised each a third primer oligonucleotide ( P3 Primer) from the following list:

• The third primer oligonucleotide (SEQ ID NO XX):
  • P3F5D-600-201 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA CTGTCCAGGGATC 3 '
  • A = 2'-deoxy-adenosine
  • C = 2'-deoxy-cytosine
  • G = 2'-deoxy-guanosine
  • T = 2'-deoxy-thymidine (thymidine)

• TMR (= Tetramethylrhodamin) in der 5' Position
  • • Das dritte Primer-Oligonukleotid (SEQ ID NO XX):
    • P3F5D-600-202 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA CCAGGGAT 3'
    • A = 2'-deoxy-Adenosin
    • C = 2'-deoxy-Cytosin
    • G = 2'-deoxy-Guanosin
    • T = 2'-deoxy-Thymidin (Thymidin)

modifications:

• TMR (= tetramethylrhodamine) in the 5 'position
  • The third primer oligonucleotide (SEQ ID NO XX):
    • P3F5D-600-202 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA CCAGGGAT 3 '
    • A = 2'-deoxy-adenosine
    • C = 2'-deoxy-cytosine
    • G = 2'-deoxy-guanosine
    • T = 2'-deoxy-thymidine (thymidine)

• TMR (= Tetramethylrhodamin) in der 5' Position
  • • Das dritte Primer-Oligonukleotid (SEQ ID NO XX):
    • P3F5D-600-203 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA TGTCCAGGGA 3'
    • A = 2'-deoxy-Adenosin
    • C = 2'-deoxy-Cytosin
    • G = 2'-deoxy-Guanosin
    • T = 2'-deoxy-Thymidin (Thymidin)

modifications:

• TMR (= tetramethylrhodamine) in the 5 'position
  • The third primer oligonucleotide (SEQ ID NO XX):
    • P3F5D-600-203 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA TGTCCAGGGA 3 '
    • A = 2'-deoxy-adenosine
    • C = 2'-deoxy-cytosine
    • G = 2'-deoxy-guanosine
    • T = 2'-deoxy-thymidine (thymidine)

• TMR (= Tetramethylrhodamin) in der 5' Position
  • • Das dritte Primer-Oligonukleotid (SEQ ID NO XX):
    • P3F5D-600-204 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA CTGTCCAG 3'
    • A = 2'-deoxy-Adenosin
    • C = 2'-deoxy-Cytosin
    • G = 2'-deoxy-Guanosin
    • T = 2'-deoxy-Thymidin (Thymidin)

modifications:

• TMR (= tetramethylrhodamine) in the 5 'position
  • The third primer oligonucleotide (SEQ ID NO XX):
    • P3F5D-600-204 TMR 5'GTACCGAAGCTCGCAGGAACTCAGAGTGTGGAGAGGACGAAAA CTGTCCAG 3 '
    • A = 2'-deoxy-adenosine
    • C = 2'-deoxy-cytosine
    • G = 2'-deoxy-guanosine
    • T = 2'-deoxy-thymidine (thymidine)

• TMR (= Tetramethylrhodamin) in der 5' Position

modifications:

• TMR (= tetramethylrhodamine) in the 5 'position

The nucleotides and nucleotide modifications are linked together with phosphodiester bonds. The 3 'end is blocked with a phosphate group to prevent possible extension by the polymerase.

The startup nucleic acid chain 1.1 (Template) was used in concentrations of 1 pmol / l. Primer 1 was used at 5 μmol / l, the controller oligonucleotide at 2 μmol / l, primer 2 at 1 μmol / l and primer 3 at 0.5 μmol / l. For amplification, 2 polymerases were in the Taq polymerase (NEB, catalog # M0273S?) In a 1-to-100 dilution and Bst 2.0 Warm Start Polymerase (NEB, Catalog # M0538S?) In a 1-to-200 dilution.

In a control reaction no P3 -Primer used. In further control reactions only BST polymerase but no Taq polymerase was used.

• • 1x Isothermal Puffer (New England Biolabs, catalog # B0537S; in einfacher Konzentration enthält der Puffer:
  • 20 mM Tris-HCI
  • 10 mM (NH4)2SO4
  • 50 mM KCl
  • 2 mM MgSO4
  • 0.1% Tween® 20
  • pH 8.8@25°C)
• • dNTP (dATP, dCTP, dTTP, dGTP), je 200 µmol/l
• • EvaGreen Farbstoff (Farbstoff wurde entsprechend Anleitung des Herstellers in Verdünnung 1:50 eingesetzt)

The further reaction conditions were:

• • 1x isothermal buffer (New England Biolabs, catalog # B0537S; in simple concentration the buffer contains:
  • 20 mM Tris-HCl
  • 10mM (NH4) 2SO4
  • 50 mM KCl
  • 2mM MgSO4
  • 0.1% Tween® 20
  • pH 8.8 @ 25 ° C)
• • dNTP (dATP, dCTP, dTTP, dGTP), 200 μmol / l each
• • EvaGreen dye (dye was used according to manufacturer's instructions in dilution 1:50)

• • 2 min 65 °C zur Aktivierung des Systems
• • 15 bzw. 30 Zyklen mit folgendem Temperatur-Zeit-Profil (Controller-Oligonukleotid-basierte Amplifikationsreaktion)
  • ◯ 2 min 55 °C
  • ◯ 2 min 65 °C
• • 20 Sekunden 95 °C zur Aktivierung des Systems
• • 30 Zyklen mit folgendem Temperatur-Zeit-Profil (Detektions-PCR)
  • ◯ 1 min 55 °C
  • ◯ 3 min 72 °C
  • ◯ 20 Sekunden 95 °C
• • 10 min 72 °C zur Vereinheitlichung der Amplifikationsprodukte
• • Schmelzkurvenanalyse

The thermal reaction conditions were chosen according to the profile:

• • 2 min 65 ° C to activate the system
• 15 or 30 cycles with the following temperature-time profile (controller oligonucleotide-based amplification reaction)
  • ◯ 2 min 55 ° C
  • ◯ 2 min 65 ° C
• • 20 seconds 95 ° C to activate the system
• • 30 cycles with the following temperature-time profile (detection PCR)
  • ◯ 1 min 55 ° C
  • ◯ 3 min 72 ° C
  • ◯ 20 seconds 95 ° C
• • 10 min 72 ° C to standardize the amplification products
• • melting curve analysis

• Erste Amplifikation:
  • (15 bzw. 30) x (2 min 55°C + 2 min 65°C) = (15 bis 30) x 4 min = 60 bis 120 min zuzüglich
• zweite Amplifikation (PCR)
  • 30 x (1 min 55°C + 3 min 72°C + 20 s 95°C) = 30 x 260 s = 130 min verfolgt. Dies entspricht einer Gesamtzeit von 190 bis 250 min.

The total amplification was typically over 45-60 cycles, ie:

• First amplification:
  • (15 or 30) x (2 min 55 ° C + 2 min 65 ° C) = (15 to 30) x 4 min = 60 to 120 min plus
• second amplification (PCR)
  • Followed 30 x (1 min 55 ° C + 3 min 72 ° C + 20 s 95 ° C) = 30 x 260 s = 130 min. This corresponds to a total time of 190 to 250 min.

• • Fluoreszenzsignal eines interkalierenden Farbstoffes (EvaGreen)
• • Schmelzkurvenanalyse von den entstandenen Amplifikationsprodukten

The successful amplification could be detected by an increase in the EvaGreen fluorescence signal over time. Analysis of the combination of controller oligonucleotide-based amplification reaction and detection PCR. The following techniques were used to analyze and evaluate the resulting amplification products:

• Fluorescence signal of an intercalating dye (EvaGreen)
• Melting curve analysis of the resulting amplification products

The first partial amplification (15 cycles or 30 cycles) leads to an accumulation of the nucleic acid chains (the first amplification fragment 1.1 ), which can be used as templates in the downstream PCR. As a result, the PCR requires fewer cycles to generate detectable levels of PCR product. By choosing the number of cycles in the two amplification phases, the amplification fraction can be adjusted to the total amplification for the two amplification reactions.

By comparison with a number of different control approaches, we show that by using a first amplification, the cycle number of the second amplification (PCR) could be reduced until a detectable signal was generated (Ct). This reduction in the number of cycles of PCR occurred only when the first amplification (controller oligonucleotide-based amplification reaction) was successful. The second amplification then proceeded from first amplification products to detection. For this reason, the downstream, classical PCR can also be called a detection PCR.

Furthermore, we show that a combination of the first and the second amplification system in one approach is possible, so that a homogeneous assay format could be performed.

Below is the presentation of results of individual reaction approaches.

52A shows a typical course of the EvaGreen fluorescence signal during the combined amplification of template M2SF5-M001-200 with primer P3F5D-600-201. On the Y-axis is the fluorescence signal of the EvaGreen dye and on the X-axis the reaction time (as cycle number) is plotted. In the cycles 1 to 15 the controller oligonucleotide-based amplification reaction is used. From cycle 15 until cycle 45 the classic PCR (detection PCR) is used. The switching time (end of cycle 15 ) is with arrow 1 marked.

arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified starting from 1 pmol / l starting concentration by BST and Taq polymerase.

arrow 3 marks a control batch containing only BST polymerase. An amplification is omitted because the heat-sensitive BST polymerase from cycle 16 denatured by the temperature profile in the detection PCR. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

arrow 4 marks another control approach which dispenses with the addition of primer P3F5D-600-201. An amplification signal is no longer observed, since without the third primer no exponential amplification in the detection PCR is possible. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

52 B shows a typical course of the EvaGreen fluorescence signal during the combined amplification of template M2SF5-M001-200 with primer P3F5D-600-202. On the Y-axis is the fluorescence signal of the EvaGreen dye and on the X-axis the reaction time (as cycle number) is plotted. In the cycles 1 to 15 the controller oligonucleotide-based amplification reaction is used. From cycle 15 until cycle 45 the classic PCR (detection PCR) is used. The switching time (end of cycle 15 ) is with arrow 1 marked.

arrow 2 marks an amplification approach in which the template M2SF5- M001 Is amplified starting from 1 pmol / l starting concentration by BST and Taq polymerase.

arrow 3 marks a control batch containing only BST polymerase. An amplification is omitted because the heat-sensitive BST polymerase from cycle 16 denatured by the temperature profile in the detection PCR. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

arrow 4 marks another control approach in which the addition of the primer P3F5D-600-202 was omitted. An amplification signal is no longer observed, since without the third primer no exponential amplification in the detection PCR is possible. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

53A shows a typical course of the EvaGreen fluorescence signal during the combined amplification of template M2SF5-M001-200 with primer P3F5D-600-203. On the Y -Axis is the fluorescence signal of the EvaGreen-dye and on the X -Axis is the reaction time (as cycle number) plotted. In the cycles 1 to 15 the controller oligonucleotide-based amplification reaction is used. From cycle 15 until cycle 45 the classic PCR (detection PCR) is used. The switching time (end of cycle 15 ) is with arrow 1 marked.

arrow 2 marks an amplification approach in which the template M2SF5- M001 Is amplified starting from 1 pmol / l starting concentration by BST and Taq polymerase.

arrow 3 marks a control batch containing only BST polymerase. An amplification is omitted because the heat-sensitive BST polymerase from cycle 16 denatured by the temperature profile in the detection PCR. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

arrow 4 marks another control approach in which the addition of the primer P3F5D-600-203 was omitted. An amplification signal is no longer observed, since without the third primer no exponential amplification in the detection PCR is possible. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

53B shows a typical course of the EvaGreen fluorescence signal during the combined amplification of template M2SF5-M001-200 with primer P3F5D-600-204. On the Y -Axis is the fluorescence signal of the EvaGreen-dye and on the X -Axis is the reaction time (as cycle number) plotted. In the cycles 1 to 15 the controller oligonucleotide-based amplification reaction is used. From cycle 15 until cycle 45 the classic PCR (detection PCR) is used. The switching time (end of cycle 15 ) is with arrow 1 marked.

arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified starting from 1 pmol / l starting concentration by BST and Taq polymerase.

arrow 3 marks a control batch containing only BST polymerase. An amplification is omitted because the heat-sensitive BST polymerase from cycle 16 denatured by the temperature profile in the detection PCR. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

arrow 4 marks another control approach in which the addition of the primer P3F5D-600-204 was omitted. An amplification signal is no longer observed, since without the third primer no exponential amplification in the detection PCR is possible. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

It turns out that a combination of controller oligonucleotide-based amplification reaction and (classical) detection PCR with each of the 4 primer-3 variants (P3F5D-600-201 to P3F5D-600-204) in homogeneous assay format succeeds. Here, the combined use of BST and Taq polymerase wirichg. Equally important is the third primer, without which no amplification signals were detected. The primer-3 variants behave quite individually, which can be recognized at the different points in time at which the amplification signal begins to stand out from the baseline. The earlier an amplification signal can be detected in the detection PCR, the more efficient is the respective primer-3 variant in the detection of previously formed controller oligonucleotide-based amplification products.

Now, we show that doubling the number of cycles of the controller oligonucleotide-based amplification reaction in the early amplification phase results in qualitatively very similar results.

54A shows a typical course of the EvaGreen fluorescence signal during the combined amplification of template M2SF5-M001-200 with primer P3F5D-600-201. On the Y-axis is the fluorescence signal of the EvaGreen dye and on the X-axis the reaction time (as cycle number) is plotted. In the cycles 1 to 30 the controller oligonucleotide-based amplification reaction is used. From cycle 31 until cycle 60 the classic PCR (detection PCR) is used. The switching time (end of cycle 30 ) is with arrow 1 marked.

arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified starting from 1 pmol / l starting concentration by BST and Taq polymerase.

arrow 3 marks a control batch containing only BST polymerase. An amplification is omitted because the heat-sensitive BST polymerase from cycle 16 denatured by the temperature profile in the detection PCR. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

arrow 4 marks another control approach which dispenses with the addition of primer P3F5D-600-201. An amplification signal is no longer observed, since without the third primer no exponential amplification in the detection PCR is possible. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

54 B shows a typical course of the EvaGreen fluorescence signal during the combined amplification of template M2SF5- M001 -200 with primer P3F5D-600-203. On the Y-axis is the fluorescence signal of the EvaGreen dye and on the X-axis the reaction time (as cycle number) is plotted. In the cycles 1 to 30 the controller oligonucleotide-based amplification reaction is used. From cycle 31 until cycle 60 the classic PCR (detection PCR) is used. The switching time (end of cycle 30 ) is with arrow 1 marked.

arrow 2 indicates an amplification approach in which the template M2SF5-M001-200 is amplified starting from 1 pmol / l starting concentration by BST and Taq polymerase.

arrow 3 marks a control batch containing only BST polymerase. An amplification is omitted because the heat-sensitive BST polymerase from cycle 16 through the temperature profile in the detection PCR is denatured. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

arrow 4 marks another control approach in which the addition of the primer P3F5D-600-203 was omitted. An amplification signal is no longer observed, since without the third primer no exponential amplification in the detection PCR is possible. Already formed amplification products from the controller oligonucleotide-based amplification reaction remain hidden below the detection threshold of EC.

Fig. 55

Shows comparison of time intervals between a combined amplification (comprising the first and the second amplification) vs. PCR alone (only the second amplification).

The first amplification was performed as shown above. In the second reaction was used as the third primer oligonucleotide (P3F5D-600-203). The batches were pipetted and reactions performed as described above. As shown above, in a combined approach, a controller-dependent amplification was first performed (in this case 15 cycles) and then switched to PCR (arrow 1 ). The time intervals were compared 1 and 2 ,

arrow 2 shows the course of the combined amplification (Bst and Taq polymerase were both in the beginning). Initially, 1 pmol / l start-up nucleic acid chain (template) was present before the start of the first amplification. The increase of the fluorescence signal occurs after a time interval 1 ,

arrow 3 shows the course of a control approach in which the Bst was omitted, so that the first amplification reaction was "shut down" (although all components of the first amplification system were in the beginning, yet lacking the right polymerase, in this case Bst 2.0 Polymerase). In the beginning, 1 pmol / l start nucleic acid chain (template) was before the start of the first amplification. The increase of the fluorescence signal occurs after a time interval 2 , Although the Taq polymerase can synthesize a product starting from the same template, the PCR alone needs more cycles for it.

arrow 4 Shows the course of a combined approach without a starting nucleic acid chain 1.1 (without matrix = NTC control).

Overall, one sees that by the proliferation of amplification fragments 1.1 during the first amplification, which is subsequently used in the PCR as starting nucleic acid chain 2.1 (Template) can serve, the generation of PCR fragments, the reaction takes place in the beginning 2 faster overall: the PCR requires fewer cycles until a detectable amount of the second amplification product ( 2.1 ) was produced. time interval 1 is shorter than time interval 2 ,

Fig. 56

Shows comparison of time intervals between a combined amplification (comprising the first and the second amplification) vs. PCR alone (only the second amplification).

The first amplification was performed as shown above. In the second reaction was used as the third primer oligonucleotide (P3F5D-600-203). The batches were pipetted and reactions performed as described above. As shown above, in a combined approach, a controller-dependent amplification was first performed (in this case 30 cycles) and then switched to PCR (arrow 1 ). The time intervals were compared 3 and 4 ,

arrow 2 shows the course of the combined amplification (Bst and Taq polymerase were both in the beginning). In the beginning, 1 pmol / l start nucleic acid chain (template) was before the start of the first amplification. Of the Increase of the fluorescence signal occurs after a time interval 3 ,

arrow 3 shows the course of a control approach in which the Bst was omitted, so that the first amplification reaction was "shut down" (although all components of the first amplification system were in the beginning, yet lacking the right polymerase, in this case Bst 2.0 Polymerase). In the beginning, 1 pmol / l start nucleic acid chain (template) was before the start of the first amplification. The increase of the fluorescence signal occurs after a time interval 4 , Although the Taq polymerase can synthesize a product starting from the same template, the PCR alone needs more cycles for it.

arrow 4 Shows the course of a combined approach without a starting nucleic acid chain 1.1 (without matrix = NTC control).

Overall, one sees that by the proliferation of amplification fragments 1.1 during the first amplification, which is subsequently used in the PCR as starting nucleic acid chain 2.1 (Template) can serve, the generation of PCR fragments, the reaction takes place in the beginning 2 faster overall: the PCR requires fewer cycles until a detectable amount of the second amplification product ( 2.1 ) was produced. time interval 3 is shorter than time interval 4 ,

Further examples

1 schematically shows the starting materials and products of an amplification comprising a first amplification ( 1A) (first part amplification) and then a second amplification ( 1 B) (second partial amplification) using components of the first amplification system and the second amplification system. The reaction starts with the starting nucleic acid 1.1 which comprises a target sequence and is used as a template to start the first partial amplification.

Components of the first amplification system are in particular: a first oligonucleotide primer ( P1.1 ), a second oligonucleotide primer ( P2.1 ), a controller oligonucleotide ( C1.1 ) and first polymerase (Pol 1.1 ), dNTPs.

During the first partial amplification ( A1.1 ) there is a specific amplification of a first amplification fragment 1.1 , comprising a first primer extension product P1.1 -Ext and a second primer extension product P2.1 -Ext. The primer extension product P1 .1-Ext occurs as a template-dependent extension of P1.1 , By the polymerase and the primer extension product 2.1 -Ext results from a template-dependent extension of the P2.1 under reaction conditions of the first partial amplification. Both primers P1.1 and P2.1 include sequence segments which can complementarily bind to the target sequence (s) such that the primers are positioned at both ends of the target sequence.

The amplification product formed during the first partial amplification 1.1 can be called start nucleic acid 2.1 for the second partial amplification ( A2.1 ) be used. During the second partial amplification becomes a second amplification fragment 2.1 amplified, comprising a third primer extension product P3 .1-Ext and a fourth primer extension product P4.1 -Ext., Wherein the primer extension product P3 .1-Ext as a template-dependent extension of P3.1 carried out, and by the polymerase and the primer extension product 4.1-Ext of the template-dependent extension of the P4.1 under reaction conditions of the second partial amplification. Both primers P3.1 and P4.1 include sequence segments which can bind complementarily to the target sequence or their complementary sequence portions. P1.1 and P3.1 Although they can complementarily bind to the controller oligonucleotide with their 3 'segments, they are not extended by the polymerases used due to the modification of the controller oligonucleotide.

2 schematically shows starting materials and products of a first partial amplification. During the first partial amplification, specific amplification of a first amplification fragment occurs 1.1 , comprising a first primer extension product P1.1 -Ext and a second primer extension product P2.1 -Ext .. The primer extension product P1.1 -Ext takes place as a template-dependent extension of P1.1 , and the primer extension product 2.1-Ext results from template-dependent extension of the P2.1 ,

The synthesis products formed during the reaction ( P1.1 -Ext and P2.1 -Ext) can form different complex forms with each other and with the controller oligonucleotide (depending on the concentration ratio and reaction conditions). Specifically, these forms can be complexes P1.1 -Ext / C1.1 and or P1.1 -Ext and / or P1.1 -Ext / C1.1 / P2.1 -Ext include.

The P1 Extract comprises a 3 'segment which includes sequence portions which are substantially complementary to one another P4.1 Primers under reaction conditions of the second partial amplification can bind. The P2 Extract comprises a 3 'segment which includes sequence portions which are substantially complementary to one another P3.1 -Primer can bind under reaction conditions of the second partial amplification.

3 schematically shows a temperature profile of the two partial amplifications ( 3A) and a schematic illustration of product accumulation (as an increase in copy number in a time-dependent manner) during the first and second partial amplifications ( 3B) ,

The course of the first partial amplification A1.1 occurs under temperature conditions which do not allow nonspecific, spontaneous strand separation in the absence of a specific controller oligonucleotide. In essence, the reaction is between temperature T1 and T2 from. At lower temperature ( T1 ) is preferably a hybridization of at least one primer of the first amplification system and its extension by the polymerase, at the second temperature ( T2 ) is preferably a separation of P1 -Ext and P2 -Ext with the help of the controller oligonucleotide. The reaction proceeds as a cyclic change in the reaction temperatures, whereby the required number of cycles can be chosen, and can influence the extent of the amplification. Here is shown schematically an increase of about 10 copies to about 10 ⁸ copies ( 3B) , Furthermore, it is shown schematically here that after completion of the first partial amplification, a proportion of products formed (amplification product 1.1 ) was transferred as an aliquot into the second partial amplification and can serve as a starting nucleic acid chain for the second partial amplification. Thus, when diluting the batch after the first partial reaction, only a partial amount of the copies formed in the first reaction is transferred to the second reaction. Also, the components of the first amplification system are diluted so that their effect on the second amplification system is reduced because of resulting low concentrations.

3A also shows schematically the course of the second partial amplification ( A2 .1), wherein the temperature conditions used are selected so that the separation of the resulting amplification products at the temperature ( T3 ) is predominantly nonspecifically due to thermal destabilization of the bonds between both strands of the synthesized complementary primer extension products and without significant involvement of controller oligonucleotide. Primer binding and extension occurs at T1 similar to the first part amplification. The reaction essentially proceeds like a PCR, with the necessary number of PCR cycles being performed to generate the desired copy number. The reactions include the binding of primers to the template, their extension to the formation of corresponding primer extension products and separation by formed strands by temperature. This results in the amplification product 2.1 , The PCR starts with an initial amount of copies of the first amplification product 1.1 comprising a target sequence of about 10 ⁵ and amplifies the amplification product 2.1 comprising the target sequence up to a copy number of about 10 ¹⁰ .

The first partial amplification proceeds under conditions which cause a sequence-dependent separation of the synthesized strands with the assistance of the controller oligonucleotide. The second partial amplification proceeds as PCR, the strand separation predominantly sequence-unspecific.

4 schematically shows a temperature profile of the two partial amplifications ( 4A) and a schematic illustration of product accumulation (as an increase in copy number in a time-dependent manner) during the first and second partial amplifications ( 4B) , The reactions are as in 3 with the difference that the components of the second amplification system will admit to the first partial amplification approach such that the number of copies of the first amplification fragment formed 1.1 the starting amount of starting nucleic acids 2.1 represents for the second partial amplification and no significant dilution of the components of the first amplification system takes place.

5 schematically shows the starting materials and products of an amplification comprising a first partial amplification ( 5A) and then a second partial amplification ( 5B) using the components of the first amplification system and the second amplification system. The reaction is essentially as in 1 shown, with the difference that during the second partial amplification, the primer P3.2 and P4.2 which do not contain complementary sequence segments to 3'-terminal segments of P1.1 -Ext or P2.1 -Ext include. Thereby can P1.1 -Ext and P2.1 -Ext products with their 3'-ends no complementary binding to the primer P3 .2 resp. P4.2 which of a polymerase (eg Pol 1.1 or pol 2.1 ). This prevents that P1.1 -Ext and P2.1 -Ext when using the primer P3 .2 and P4.2 be excessively extended under reaction conditions of the first partial amplification. During the second partial amplification training of the complete P3.1 -Ext- and P4.1 -Ext products during the cyclic synthesis and their multiplication. The use of such primer combinations (Set 1 full P1.1 and P2.1 and set 2 full P3.1 and P4.1 ) allow a design of homogeneous reactions in which both primer sets are provided at the beginning of amplification in the same reaction approach.

6 schematically shows a temperature profile of both partial amplifications ( 6A) and a schematic illustration of product accumulation (as an increase in copy number in a time-dependent manner) during the first and second partial amplifications ( 6B) ,

The figure schematically shows a temperature profile in which both partial amplifications are performed immediately after each other (both primer sets are present at the beginning of the reaction). Schematically, it is shown that during the first amplification, the increase in the number of copies of the amplification product 1.1 from about 10 to about 10 ⁴ . These products comprise a target sequence and serve as a starting nucleic acid 2.1 for the second amplification. During the second amplification, there is a further increase in the number of amplified target sequences starting from approximately 10 ^{4 of} the amplification products 1.1 to about 10 ¹⁰ resulting amplification products 2.2 ,

7A schematically shows a temperature profile of the two partial amplifications. The expiration of temperature changes in 7A matches those in 3 described: The sequence of the first partial amplification A1 .1 occurs under temperature conditions which do not allow nonspecific, spontaneous strand separation in the absence of a specific controller oligonucleotide. In essence, the reaction is between temperature T1 and T2 from. At lower temperature ( T1 ) is preferably a hybridization of at least one primer of the first amplification system and its extension by the polymerase, at the second temperature ( T2 ) is preferably a separation of P1 -Ext and P2 -Ext with the help of the controller oligonucleotide. During the course of the reaction, the amplification of the first amplification products takes place 1.1 , which is used as starting nucleic acid 2.1 can be used in the second part amplification. The reaction proceeds as a cyclic change in the reaction temperatures, whereby the required number of cycles can be chosen, and can influence the extent of the amplification. 7A also shows schematically the course of the second partial amplification ( A2.1 ) which takes place immediately after the first partial amplification, wherein the temperature conditions used are chosen so that the separation of the resulting amplification products at the temperature ( T3 ) predominantly nonspecifically by thermal destabilization of the bonds between both strands of the synthesized complementary primer extension products and without substantial involvement of the controller oligonucleotide. Primer binding and extension occurs at T1 similar to the first part amplification. The reaction essentially proceeds like a PCR, with the necessary number of PCR cycles being performed to generate the desired copy number. The reactions include binding of the primers to the template, their extension to formation of corresponding primer extension products, and separation of formed strands by temperature. This results in the amplification product 2.1 , The first partial amplification proceeds under conditions which induce a sequence-dependent separation of synthesized strands with the assistance of the controller oligonucleotide. The second partial amplification proceeds as PCR, the strand separation predominantly sequence-unspecific.

7B shows a first and a second amplification as in FIG 7A , Both reactions are separated by a further temperature step, here referred to as "temperature switching step", which involves raising the temperature to about T3 for a certain period of time. During this step, for example, an inactivation of the polymerase of the first partial amplification can take place and / or an activation of the polymerase of the second partial amplification and / or an activation of thermolabile primers. With such a step, there is thus also a change in the activity of the individual components of the amplification systems.

8th schematically shows a temperature profile of both partial amplifications. The procedure first partial amplification corresponds to those as in 7A described. The sequence of the second partial amplification is divided here into two phases. During the first phase ( A2.1 phase 1 ) there is a temperature change between T1 and T3 , While T1 At least one primer of the second amplification system can be attached to its corresponding complementary position in the amplification product 1.1 bind and be extended. By using lower temperatures, it is also possible to use primers with relatively short 3 'segments which can bind complementary (as in US Pat 5 shown). phase 1 may include several cycles, with the initial amplification products 2.1 which comprise complementary segments which are longer and thus capable of binding primers at higher temperatures. This allows use of higher temperatures in the phase 2 for primer binding and primer extension step (here as cyclic extensions between T2 and T3 shown). By increasing the temperature, for example, a generation of side reactions can be counteracted.

8B Here is shown schematically that the temperature during the A2.1 phase 1 continue on temperature T4 is lowered so that, for example, the primer of the set 2 can bind. The use of temperature T4 in phase 1 For example, by using at least primer of the set 2 comprising at least one mismatch to complementary sequence segments of the first amplification products conditional. Low temperature ( T4 ) an initial generation of primer extension products starting from P3.1 and or P4.1 support. After this initial generation, the temperature of the step can be increased with primer binding and primer extension (phase 2 ), since fully complementary primer binding sites are present.

9A schematically shows a temperature profile of the two partial amplifications. The sequence of first partial amplification takes place under a uniform temperature, here at T2 , All necessary steps of the first partial amplification run at this temperature. Subsequently, for example, a temperature increase (temperature-switching-Schitt) follow (as in 7B described). Subsequently, a PCR reaction is carried out as a second partial amplification.

The first partial amplification proceeds at a uniform temperature, which favors a sequence-dependent strand separation with the assistance of controller oligonucleotide.

9B schematically shows a further temperature profile in the first partial amplification, which includes both an isothermal phase (at the beginning) and a later cyclic phase. Here, too, the first partial amplification proceeds at temperatures which favor a sequence-dependent strand separation with the assistance of the controller oligonucleotide.

10A schematically shows a temperature profile of the two partial amplifications. The second partial amplification in phase 2 involves the use of another temperature T5 which is higher than T2 , This temperature is between about 68 ° C and 82 ° C. The use of temperature T5 may be the binding of the controller oligonucleotide to the P3.1 -Ext weaken, and thus the influence of this binding on extension of P4.1 reduce, so that extension of P4.1 possibly more efficient.

10B schematically shows a temperature profile of both partial amplifications. During the second part amplification takes place in the phase 1 an initial generation of the second amplification products. Using the primer 3.1 and 4.1 which are able to withstand higher temperatures ( T5 ) to bind to their complementary positions and to be extended by polymerase, the temperature in the second phase between T5 and T3 to be changed. By choosing the reaction conditions, one can influence the formation of intermediates or end products. For example, using longer P3.1 and P4.1 (eg 30-40 nucleotides long) in combination with a higher temperature at the primer binding step and primer extension step in the second partial amplification (eg at T2 and or T5 ) are favored P3.1 -Ext and P4.1 -Ext accumulates.

11 schematically shows a temperature profile of both partial amplifications. The transition phase between both partial amplifications can be designed differently. In 11A a continuous increase in temperature is shown at the end of the first partial amplification. In 11B a cyclic decrease in temperature is shown at the end of the first partial amplification. Thus, both partial amplifications may have partial overlaps in reaction sequences.

In the figures 12 - 19 the relations between individual components of the first amplification system are shown.

In the 20 - 26 Figures are shown between components of the first amplification system and the second amplification system. 20A shows schematically the generation of the first amplification products (comprising P1.1 -Ext and P2.1 -Ext) starting from a starting nucleic acid 1.1 (which is schematically added here as the nucleic acid to be amplified in the reaction). 20B schematically shows generation of intermediates using the primer P3 .1 and P4.1 and P1.1 -Ext and P2.1 -Ext of the first partial amplification as templates arise ( P3.1 -Ext part1 and P4.1 -Ext part 1 ). These intermediates may become complete in subsequent cycles by re-primer binding and extension P3.1 -Ext and P4.1 -Ext using P3.1 -Ext part1 and P4.1 -Ext part 1 be converted as matrices. The choice of conditions can influence the formation of intermediates or end products. For example, using longer P3.1 and P4.1 (eg, 30-40 nucleotides long) in combination with a higher temperature at the primer binding step and primer extension step in the second partial amplification (eg at T2 and or T5 ) are favored P3.1 -Ext and P4.1 -Ext accumulates.

The figures 27 to 35 schematically show examples of arrangements of sequence segments of a controller oligonucleotide ( C1 .1), the first primer ( P1 .1) and the third prime ( P3 .1) and a second primer extension product ( P2 .1-Ext). The P2 .1-Ext includes in the 3 'segment a for P1.1 complementary sequence and can be used as a template for the generation of P1.1 Text in the first part amplification. Furthermore, this includes P2.1 -Ext another complementary segment for 3'-segment of the P3.1 , which to the P2.1 -Ext bind and can be extended by polymerase, so that in the second partial amplification P3.1 -Ext can be generated.

The 27 to 30 summarize examples of arrangements of segments in the controller oligonucleotide and P1.1 and P3.1 together. P3.1 can bind with its 3 'segment to the controller oligonucleotide complementary. This binding takes place in a sequence segment of the controller oligonucleotide, which is referred to here as the fourth region. Depending on how the positioning of the P3.1 This fourth area may accordingly have different localization with respect to areas one, two or three. In one embodiment, the fourth region is within the second region (FIG. 27 In another embodiment, the fourth region lies at the transition between the regions two and three ( 29 In another embodiment, the fourth region lies in the third region of the controller oligonucleotide ( 30 ). Preferably, the presence of the controller oligonucleotide should not lead to unwanted formation of by-products. Most importantly, primer binding to the controller oligonucleotide should not cause the polymerase to undergo a control oligonucleotide using the controller oligonucleotide P1.1 Extension or P3.1 Extension catalyzes. This is generally accomplished by using nucleotide modifications in the controller oligonucleotide, eg, by using 2'-O-alkyl modifications. Such modifications are preferably employed in sequence segments of the controller oligonucleotide which comprise complementary sequences at least to the 3 'segment of the respective primer. Furthermore, such modifications may go beyond the sequence segment complementary to a primer, for example, these sequence segments may comprise positions from about minus 10 to about plus 10 nucleotides, respectively, the position of 3 'end of a respective primer. The sequence segments having such nucleotide modifications are referred to herein as "second blocking moiety" which comprises the complementary sequence segments to the first primer (FIG. P1 .1) include ( 27 - 31 ), or as "fourth blocking unit" which complementary sequence segments to the third primer ( P3 .1) include ( 27 - 31 ). Such modification of controller oligonucleotides thus prevents the polymerases from initiating a template-dependent primer extension upon primer binding to the controller oligonucleotides. The composition of the nucleotide modifications may be the same or different in the second and fourth blocking units. The length of the respective sequence segments, which are combined into a second or fourth blocking unit, may also be the same or different for the respective blocking units. The location of the respective blocking units depends on the position of the respective potential binding sites of the primers on the controller oligonucleotide ( 27 - 31 ). A possible composition of the second blocking unit for the first primer is shown in detail. The composition of the fourth blocking unit can be designed analogously on the basis of the second blocking unit. In a preferred embodiment, the third region of the controller oligonucleotide consists entirely of 2'-O-alkyl modifications of nucleotides. In a further preferred embodiment, the third region of the controller oligonucleotide in its 3 'segment (eg, nucleotide positions from 1 to 20 subsequent to the second region of the controller oligonucleotide) consists entirely of 2'-O-alkyl modifications of nucleotides. In another preferred embodiment, the second region of the controller oligonucleotide consists entirely of 2'-O-alkyl modifications of nucleotides. In another preferred embodiment, the second region of the controller oligonucleotide and the third region of the controller oligonucleotide are in its 3 'segment (positions 1 to 20 subsequent to the second region) completely from 2'-O-alkyl modifications of nucleotides. Thus, it is not absolutely necessary to provide individual segments with modifications separately, complete sequence segments of the controller oligonucleotide can be provided by complementary nucleotides, for example with 2'-O-alkyl modification.

Due to a lack of modification in complementary sequence segments of the primer extension products (which are generated as a result of an enzymatic activity of polymerases in a template-dependent manner), primers can be recognized and extended upon complementary binding to such sequence segments of polymerases.

The 35 to 39 schematically show examples of arrangements and potential combinations comprising primer set 1 ( P1.1 and P2.1 ) as well as primer set 2 ( P3.1 and P4.1 ). It can be one of the primers of primer set 1 with one of the primers of Set 2 be identical (eg P4.1 corresponds to P2.1 in 35 - 37 ). Especially when using a dilution of the reaction mixture after the first partial amplification and adding the components of the second amplification system after the first partial amplification has elapsed, the primers of the second primer set may be of their length from the primer set 1 distinguish ( 38 ). Preference is given to primer 3 Used structures which comprise only in their 3 'segment a complementary sequence to the controller oligonucleotide. The composition of the 5 'segment of the third primer preferably does not comprise any complementary sequence segments to the controller oligonucleotide ( 39 ).

The 40 to 43 show schematically the interaction of components in a first partial amplification.

40 describes components of in 42 - 44 represented structures.

41 schematically shows the strand displacement mechanism.

42 shows schematically the interaction of the structures in strand displacement.

43 shows schematically the interaction of the structures during the nucleic acid amplification by on the one hand the amplification of the first primer oligonucleotide and the second primer oligonucleotide and further the action of the controller oligonucleotide and the resulting strand displacement.

44 shows results from Example 1.

45 shows results from Example 2.

47 - 51 shows results from Example 3.

52 - 56 shows results from Example 4.

57 - 58 schematically shows a preparation of a starting nucleic acid chain 1.1

59 - 61 schematically shows the topography of components of the first amplification system.

The start nucleic acid 1.1 comprises (in the 5'-3 'direction) the following segments: M1 .Y, M1.5 . M1.4 . M1.3 . M1.2 . M1.1 . M1 .X.

The first primer oligonucleotide comprises (in the 5'-3 'direction) the following segments: the first region ( P1.1.1 ) and the second area ( P1.1.2 ).

The controller oligonucleotide ( C1.1 ) comprises (in the 5'-3 'direction) the following segments: the third region ( C1.1 .3), the second area ( C1.1.2 ), the first area ( C.1.1.1 );

The controller oligonucleotide ( C1 .2) comprises (in the 5'-3 'direction) the following segments: the third region ( C1.2.3 ), the second area ( C1 .2.2), the first area ( C.1.2.1 );

The second primer P2.1 comprises (in the 5'-3 'direction) the following segments: P2.1.1

The second primer P2.2 comprises (in the 5'-3 'direction) the following segments: P2.2.1

The second primer P2.3 comprises (in the 5'-3 'direction) the following segments: P2.3.1

The first primer extension product ( P1.1 -Ext) comprises (in the 5'-3 'direction) the following segments: P1.1E6 . P1.1E5 . P1.1E4 . P1.1E3 . P1.1E2 . P1.1E1 ,

The second primer extension product ( P2 .1-Ext) comprises (in the 5'-3 'direction) the following segments: P2.1E5 . P2.1E4 . P2.1E3 . P2.1E2 . P2.1E1 ,

The primer extension products preferably obtained during the first amplification P1 .1-Ext and P2.1 -Ext can form complementary double strand and together make up the second amplification fragment 1.1 which is used as starting nucleic acid 2.1 can serve for the second amplification.

In which P1.1.1 complementary to M1.1 can bind predominantly complementary and can be extended by polymerase. P2.1.1 at P1.1E1 can bind complementary and can be extended. P1.1 -Ext and P2.1 -Ext represent the nucleic acid to be amplified, which are used as starting nucleic acid 2.1 for the second amplification. P2.1 . P2.2 , and P2.3 represent variants of second primer and result in equal amplification fragments.

60 schematically shows topography of the second amplification system:

The third primer oligonucleotide comprises (in the 5'-3 'direction) the following segments: P3.1.2 . P3.1.1 , in which P3.1.2 not complementary to starting nucleic acid 1.1 or to P2.1 -Ext is. P3.1.1 is essentially complementary to P2.1E1 of P2.1 -Ext.

The fourth primer oligonucleotide comprises (in the 5'-3 'direction) the following segments: P4.1.2 . P4.1.1 , in which P4.1.2 not complementary to the complementary strand of the starting nucleic acid 1.1 or to P1.1 -Ext is. P4.1.1 is essentially complementary to P1.1E1 of P1.1 -Ext.

The primer extension products preferably obtained during the second amplification P3 .1-Ext and P4.1 -Ext can form complementary double strand and together make up the second amplification fragment 2.1 represents.

P3.1 -Ext comprises (in the 5'-3 'direction) the following segments: P3.1E7 . P3.1E6 . P3.1E5 . P3.1E4 . P3.1E3 . P3.1E2 . P3.1E1 , The segments P3.1. E5 to P3.1E3 are identical in their sequence P1.1E4 to P1.1E2 ,

P4.1 -Ext comprises (in the 5'-3 'direction) the following segments: P4.1E7 . P4.1E6 . P43.1E5 . P4.1E4 . P4.1E3 . P43.1E2 . P4.1E1 , The segments P4.1.E5 to P4.1E3 are identical in their sequence P2.1E4 to P2.1E2 ,

61 shows variants of variants of the third primer ( P3 .1, P3.2 . P3.3 ) and the fourth primer ( P4.1 . P4.2 . P4.3 ), wherein the 3 'segment of the respective primer may be shifted in relation to each other.

62 - 63 schematically shows possible localization regions for oligonucleotide probes:

D1 - is at the 5'-end of the P4.1 Text isolated. Therefore, for example, primers with probe function can be used, eg Scorpion primer, Lux primer. Wherein primers are similar in their 3 'segments P4.1 are constructed.

D4 - is at the 5'-end of the P3.1 Text isolated. Therefore, for example, primers with probe function can be used, eg Scorpion primer, Lux primer. Wherein primers are similar in their 3 'segments P3.1 are constructed.

areas D2 and D3 lie in middle segments of the P3.1 -Ext or P4.1 -Ext. Thus, for example, hybridization probes (eg, molecular beacons, Taqman probes (5'-3 'nuclease cleavable probes, or 2-oligonucleotide probes with FRET pairs) can be located here.

D2 Range is particularly well suited for potential probe localization (in P3.1 -Ext or in P4.1 -Ext) because this area does not overlap with controller oligonucleotide. Therefore, probes can be used especially in homogeneous assays in this area. This area includes the 3 'segment of the third primer extension product. Probes in the area D3 can also be used both for P3.1 -Ext as well for P4.1 -Ext be provided. Im in realtiv high concentrations of C1.2 (Controller, for example, 0.5 mol / l to 5 .mu.mol / l, eg in the context of a homogeneous assay) must be taken into account, however, that such probes may possibly interact with controller or can be displaced by the controller. At relatively low concentrations of controller (eg in the range less than 1 nmol / l), this interaction with the controller can be neglected (eg when diluting the first amplification before the second amplification).

64 schematically shows localization of probe segments which are predominantly complementary to the third (and possibly the first) primer extension product ( P3.1 -Ext and P1.1 -Ext) can bind. Various embodiments show that there are several possibilities for potential probe localizations.

65 schematically shows localization of probe segments which are predominantly complementary to the fourth (and possibly the second) primer extension product (FIG. P4 .1-Ext and P2.1 -Ext) can bind. Various embodiments show that there are several possibilities for potential probe localizations.

66 - 67 schematically shows some embodiments of probes:

Probes with localization in D1 : S3.1 . S3.2 . S3.3 , The probes include a fluorescent reporter (R) / fluorescent quencher (Q) pair, which when hybridized to corresponding segments of primer extension products (reporter / quencher spatial separation) results in signal enhancement. probe S3 .1 and S3.3 can be extended as primers, resulting in primer extension products starting from primers with probe function ( S3.1 -Ext) and S3.3 .Ext in 67 ).

In 68 - 71 schematically show some embodiments of oligonucleotide probes, as are widespread: S3.4 represents a 5'-3 'nuclease cleavable probe ("Taqman probe"). This probe, after its hybridization to the P3.1 -Ext can be cleaved by a Taq polymerase, with simultaneous P4.1 Extension takes place.

probe S3.6 represents a probe system in which 2 oligonucleotides are attached to the P3.1 -Ext can bind / need. An oligonucleotide is the probe S3.6 with fluorescence reporter (R), the other probe is the controller oligonucleotide C1.2 which comprises a donor fluorophore in its 5 'region. With simultaneous binding of both oligonucleotides P3.1 -Ext creates a sufficient spatial proximity (below 25NT), so that FRET can take place, which leads to the signal.

probe S3.7 schematically represents a probe with self-complementary portions ("Molecular Beacon"). In the non-bound state, the signal from the fluorescence reporter (R) is preferably quenched by fluorescence quencher (Q) under selected reaction conditions. Upon complementary binding, the structure unfolds, resulting in spatial separation of the R from Q, which usually results in signal increase.

72 - 74 schematically shows embodiments of localization of a target sequences 1 - 3 in the starting nucleic acid 1.1 , an amplification fragment 1.1 ( P1.1 -Ext and P2.1 -Ext), as well as the second Amplifikationsfragment 2.1 ( P3.1 -Ext and P4.1 -Ext).

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• EP 2017/071011 [0137]
• EP 16185624 [0137]
• US 5210015 [0180]

Cited non-patent literature

• Scalice et al J. Immunol. Methods, 1994, p. 147, Lyamichev et al PNAS, 1999, p. 6143 [0176]

Claims (10)

Method for amplifying a nucleic acid ( 60 ) comprising the steps of: a) a first amplification comprising the steps of: - providing a starting nucleic acid 1.1 comprising a target sequence - hybridizing a first primer oligonucleotide (P1.1) to a nucleic acid to be amplified with a target sequence (either start Nucleic acid 1.1 or P2.1-Ext), wherein the first primer oligonucleotide (P1.1) comprises the following regions: a first region (P1.1.1), the sequence-specific to a region of the nucleic acid to be amplified (P2.1E1) • a second region (P1.1.2), which connects to the 5 'end of the first region or is connected via a linker, wherein the second region can be bound by a controller oligonucleotide and for the polymerase used the selected reaction conditions remains essentially unkopiert; Extension of the first primer oligonucleotide (P1.1) by a first polymerase to obtain a first primer extension product (P1.1-Ext) which comprises a synthesized region in addition to the first primer oligonucleotide (P1.1) (P1. 1E1 to P1.1E4) which is substantially complementary to the nucleic acid to be amplified or to the target sequence, wherein the first primer extension product and the nucleic acid to be amplified are present as a double strand; Binding of a controller oligonucleotide (C1.2) to the first primer extension product (P1.1-Ext), wherein the controller oligonucleotide (C1.2) comprises the following ranges: a first region (C1.2.1), the a second region (C1.2.2) which is complementary to the first region of the first primer oligonucleotide (P1.1.1); third region (C1.2.3) that is substantially complementary to at least a portion of the synthesized region of the first primer extension product (P1.1E4); and wherein the controller oligonucleotide (C1.1) does not serve as template for primer extension of the first primer oligonucleotide (P1.1), and the first controller oligonucleotide (C1.1) to the first primer extension product (P1.1E6 P1.1E5, P1.1E4) by displacing the region (P2.1E1, P2.1E2) which is complementary to this region, of the nucleic acid to be amplified; Hybridizing a second primer oligonucleotide (P1.1) to the first primer extension product, wherein the second primer oligonucleotide (P2.1) comprises a region (P2.1.1) that is sequence-specifically to the synthesized region of the first primer extension product Extension of the second primer oligonucleotide (P.2.1) by the first polymerase to obtain a second primer extension product (P2.1-Ext) which is next to the second primer oligonucleotide (P2.1 ) comprises a synthesized region (P2.1E4 to P2.1E1) substantially identical to the nucleic acid to be amplified or to the target sequence, the first Primer extension product (P1.1-Ext) and the second primer extension product (P2.1) form a first double-stranded amplification product; and - steps of the first amplification are repeated until the desired amount of first amplification products has been synthesized. b) a second amplification comprising the steps of: hybridizing a third primer oligonucleotide (P3.2) to the second and / or the fourth primer extension product (P1.1-Ext. or P4.1-Ext), the third Primer oligonucleotide (P3.2) comprising a first region (P3.1.1) capable of sequence-specific binding to a region of the second primer extension product (P2.1E1) and the fourth primer extension product (P4.1E2), - extension of the third primer oligonucleotide (P3.2) by a second polymerase to obtain a third primer extension product (P3.2-Ext) which contains, in addition to the third primer oligonucleotide (P3.2), a synthesized region (P3.1E5 to P3. 1E2 or P3.1E5 to P3.1E1) which is substantially complementary to the second primer extension product (P2.1-Ext) or to the target sequence, the second primer extension product (P2.1) and the third primer Extension product (P3.1) is present as a double strand; Hybridizing a fourth primer oligonucleotide (P4.2) to the first primer extension product (P1.1-Ext) and / or to the third primer extension product (P3.1-Ext) wherein the fourth primer oligonucleotide (P4. 2) comprises a first region (P4.1.1) which can sequence-specifically bind to the synthesized region of the first primer extension product (P1.1E1) and the third primer extension product (P3.1E2), - extension of the fourth primer oligonucleotide (P4. 2) by the second polymerase to obtain a fourth primer extension product (P4.2-Ext) comprising a synthesized region (P4.1E5 to P4.1E2 or P4.1E5 to P4.1E1) in addition to the fourth primer oligonucleotide, which is substantially complementary to the first primer extension product (P1.1-Ext) or identical to the target sequence, the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.1-Ext) being double stranded available; Separation of the double strand from the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.2-Ext) and the double strand from the second primer extension product (P2.1-Ext) and the third primer Extension product (P3.1-Ext) and the double strand from the fourth primer extension product (P4.1-Ext) and the third primer extension product (P3.1-Ext); Hybridizing the third primer oligonucleotide (P3.2) to the fourth primer extension product (P4.2-Ext) and extension of the third primer oligonucleotide (P3.2) by the second polymerase; and - hybridizing the fourth primer oligonucleotide to the third primer extension product (P3.2-Ext) and extending the fourth primer oligonucleotide (P4.2) by the second polymerase. Method according to Claim 1 , characterized in that the steps of the first amplification are repeated until the first amplification product is present in a copy number in the range of 10 to 100,000,000,000, in particular from 100 to 1,000,000,000, in particular from 1,000 to 100,000,000. Method according to Claim 1 or 2 , characterized in that the third primer oligonucleotide and / or the fourth primer oligonucleotide have a second region (P3.1.2 or P4.1.2) which adjoins the 5 'end of the first region, the second region can not bind complementarily to the first primer extension product (P1.1-Ext) or to the second primer extension product (P2.1-Ext). Method according to one of the preceding claims, characterized in that the separation of the double strand from the first primer extension product (P1.1-Ext) and the fourth primer extension product (P4.2-Ext) and the double strand from the second primer extension product (P2.1-Ext) and the third primer extension product (P3.1-Ext) and the double strand from the fourth primer extension product (P4.1-Ext) and the third primer extension product (P3.1-Ext) thermal denaturation is carried out, in particular at a temperature in the range of 85 ° C to 105 ° C. Method according to one of the preceding claims, characterized in that the first polymerase and the second polymerase are identical. Method according to one of the preceding claims, characterized in that - the first primer oligonucleotide (P1.1) and the third primer oligonucleotide (P3.2) are substantially identical; and / or - the second primer oligonucleotide (P2.1) and the fourth primer oligonucleotide (P4.2) are substantially identical. Method according to one of the preceding claims, characterized in that the first amplification and the second amplification are carried out in the same reaction mixture. Method according to Claim 7 , characterized in that the second polymerase, the third primer oligonucleotide (P3.1) and / or the fourth primer oligonucleotide (P4.1) can be activated, and / or the controller oligonucleotide (C1.1) can be deactivated. Method according to one of the preceding claims, characterized in that a first labeled with a fluorescent dye oligonucleotide probe can bind to the third primer extension product complementary and the signal of the fluorescent dye is detected. Method according to one of the preceding claims, characterized in that a first labeled with a fluorescent dye oligonucleotide probe can bind to the fourth primer extension product complementary and the signal of the fluorescent dye is detected.

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