HK1142928B - Nucleic acid amplification in the presence of modified randomers - Google Patents

Description

Nucleic acid amplification in the presence of modified random oligonucleotides

The present invention relates to the field of template-dependent polymerase catalyzed primer extension reactions. In particular, the present invention provides a novel method for nucleic acid amplification by performing Polymerase Chain Reaction (PCR). More specifically, the present invention provides a novel method for performing hot start PCR characterized by avoiding non-specific primer dimer amplification.

Prior Art

A major problem with nucleic acid amplification and more particularly with PCR is the generation of non-specific amplification products. In many cases, this is due to non-specific oligonucleotide priming and subsequent primer extension events prior to the actual thermocycling procedure itself, since thermostable DNA polymerases are also moderately active at ambient temperature. For example, amplification products due to eventual, by chance, primer dimerization and subsequent extension are often observed. To overcome this problem, it is well known in the art to perform so-called "hot start" PCR, in which one of the components necessary for the amplification reaction is separated from the reaction mixture, or kept in an inactive state until the temperature of the reaction mixture is first raised. Because polymerases are unable to function under these conditions, there is no primer extension during the period of time when primers can bind non-specifically. To achieve this effect, several methods have been applied:

a) physical separation of DNA polymerases

Physical separation can be obtained, for example, by a barrier of solid wax that separates the compartment containing the DNA polymerase from the compartment containing most of the other reagents. During the first heating step, the wax then automatically melts and mixes the fluid compartments (Chou, Q., et al, nucleic acids Res20(1992)1717-23, US5,411,876). Alternatively, the DNA polymerase is affinity immobilized on a solid support prior to the amplification reaction and released into the reaction mixture only by heat-mediated release (Nilsson, j., et al, Biotechniques22(1997) 744-51). However, both of the 2 methods are time consuming and inconvenient to perform.

b) Chemical modification of DNA polymerases

For this type of hot start PCR, the DNA polymerase is reversibly inactivated as a result of chemical modification. More precisely, thermolabile blocking groups are introduced into taq dna polymerase, which renders the enzyme inactive at room temperature (US5,773,258). These blocking groups are removed during the pre-PCR step at high temperatures, thereby allowing the enzyme to become activated. Such thermolabile modification can be obtained, for example, by coupling citraconic anhydride or aconitic anhydride (Aconitricenhydride) to a lysine residue of the enzyme (U.S. Pat. No. 5,677,152). Enzymes carrying such modifications are currently commercially available as Amplitaqgold (Moretti, T., et al, Biotechnicques 25(1998)716-22) or FastStartDNA polymerase (Roche molecular biochemicals). However, the introduction of blocking groups is a chemical reaction that occurs arbitrarily on all sterically available lysine residues of the enzyme. Thus, the reproducibility and quality of chemically modified enzyme preparations may vary and may be difficult to control.

c) Recombinant modification of DNA polymerases

Cold sensitive mutants of Taq polymerase have been prepared by genetic engineering. These mutants differ from the wild-type enzyme in that they lack the N-terminus (US6,241,557). In contrast to native or wild-type recombinant Taq polymerase, these mutants are completely inactive below 35 ℃ and can therefore be used in some cases to perform hot start PCR. However, the N-terminally truncated cold-sensitive mutant forms require low salt buffer conditions, have a lower processivity compared to the wild-type enzyme and can therefore only be used for amplification of short target nucleic acids. Furthermore, since the truncated form lacks 5 '-3' exonuclease activity, it cannot be used in real-time PCR experiments based on the TaqMan detection format.

d) DNA polymerase inhibition via nucleic acid additives

Extension of non-specifically annealed primers has been shown to be inhibited by the addition of short double stranded DNA fragments (Kainz, P., et al, Biotechnicques 28(2000) 278-82). In this case, primer extension is inhibited at a temperature below the melting temperature of the short double-stranded DNA fragment, independent of the sequence of the competitor DNA (completeror DNA) itself. However, it is unknown to what extent the excess amount of competitor DNA affects the yield of the nucleic acid amplification reaction.

Alternatively, oligonucleotide aptamers with specific sequences that result in defined secondary structures may be used. Such aptamers have been selected for very high affinity for DNA polymerase using SELEX technology (US5,693,502, Lin, y. and Jayasena, s.d., JMolBiol271(1997) 100-11). The presence of such aptamers within the amplification mixture prior to the actual thermocycling process itself again leads to high affinity binding to the DNA polymerase and thus thermolabile inhibition of its activity (US6,020,130). However, due to the selection process, all aptamers available so far can only be used in combination with one specific kind of DNA polymerase.

e) TaqDNA antibodies

An alternative approach to achieve thermolabile inhibition of TaqDNA polymerase is to add monoclonal antibodies raised against the purified enzyme (Kellogg, D.E., et al, Biotechnology 16(1994) 1134-7; Sharkey, D.J., et al, Biotechnology (NY)12(1994) 506-9). Like oligonucleotide aptamers, antibodies bind to taq dna polymerase with high affinity at ambient temperature in an inhibitory manner (US5,338,671). The compound is decomposed in a preheating step before the thermocycling process itself. This leads to a significant time-consuming extension of the amplification as a whole, especially if the protocol for rapid thermal cycling is applied (WO 97/46706).

US5,985,619 discloses a specific embodiment for performing PCR using a hot start antibody, wherein in addition to Taq polymerase, exonuclease III, e.g. from e.coli (e.coli), is added as a supplement to the amplification mixture to digest non-specific primer dimer intermediates. As disclosed above, exonuclease III recognizes double-stranded DNA as a substrate, such as, for example, a target/primer or target/primer extension product hybrid. Digestion occurs by cleavage of the phosphodiester bond at the 5 'end of the 3' terminal deoxynucleotide residue. Since this type of exonuclease is active at ambient temperatures, all non-specifically annealed primers and primer extension products are digested. This results in some embodiments in an even enhanced specificity of the amplification reaction. However, digestion of non-specific primers depending on the duration of the pre-incubation time may result in a considerable and uncontrolled reduction of primer concentration, which in turn may affect the amplification reaction itself.

f) Use of modified primers alone or in combination with exonucleases

EP0799888 and GB2293238 disclose the addition of 3' blocked oligonucleotides to PCR reactions. Due to the 3' block, these oligonucleotides cannot act as primers. The blocked oligonucleotides are designed to compete/interact with the PCR primers, which results in a reduction of non-specific products.

Another alternative is the use of thiosulphate oligonucleotide primers in combination with exonuclease III in the PCR reaction mixture (EP 0744470). In this case, 3' exonucleases, which typically accept double-stranded as well as single-stranded DNA substrates, degrade duplex artifacts such as primer dimers and carry-over (carryover) amplicons, while leaving the single-stranded amplification primers undegraded. Similarly, the use of primers with substantially modified 3' ends and template-dependent removal via E.coli endonuclease IV has been suggested (US5,792,607).

A specific embodiment of the general idea is found in EP 1275735. Its specification discloses compositions for performing nucleic acid amplification reactions comprising (i) a thermostable DNA-polymerase, (ii) a thermostable 3 ' -5 ' exonuclease, and (iii) at least one primer for nucleic acid amplification having a modified 3 ' terminal residue that is not extended by the thermostable DNA-polymerase, as well as methods for performing PCR reactions using such compositions.

However, a major drawback of the disclosed alternative is the need for modified primers for each PCR reaction, which leads to a need for increased costs for increasing each individual assay.

g) Other PCR additives

Other organic additives known in the art, such as DMSO, betaines and formamides (WO 99/46400; Hengen, P.N., trends biochem Sci22(1997) 225-6; Chakrabarti, R. and Schutt, C.E., nucleic acids Res29(2001)2377-81) result in improved amplification of GC-rich sequences, rather than prevention of primer dimer formation. Similarly, heparin is speculated to stimulate in vitro run-on transcription presumably through the removal of proteins such as histones to make chromosomal DNA accessible (Hildebrand, C.E., et al, Biochimica tBiophysica acta477(1977) 295-311).

It is also known that the addition of single-stranded binding proteins (US5,449,603) or tRNA (Sturzenbaum, S.R., Biotechniques27(1999)50-2) results in non-covalent binding of these additives to the primers. This binding is disrupted when heating during PCR. It was also found that the addition of DNA helicase prevents random annealing of the primers (Kaboev, O.K., et al, bioorg Khim25(1999) 398-400). In addition, polyglutamic acid (WO00/68411) can be used in several cases to inhibit the polymerase activity at low temperatures.

Furthermore, it is known that polyanionic polymerase inhibitors can control the activity of thermostable DNA polymerases depending on the applied incubation temperature. US6,667,165 discloses a hot start embodiment characterized in that an inactive polymerase-inhibitor complex is formed at a temperature below 40 ℃. At 40 ℃ to 55 ℃, the inhibitor competes with the template DNA for binding to Taq polymerase, while at temperatures above 55 ℃, the inhibitor leaves the polymerase active site. However, when primers with lower annealing temperatures are used, inhibitors tend to reduce the obtainable product yield.

h) Magnesium chelation

Since thermostable polymerases have long been known to be active only in the presence of Mg2+ cation, chelation of magnesium prior to initiation of the thermocycling protocol has been attempted to avoid false priming and non-specific primer extension. As disclosed in US6,403,341, Mg2+ may be present in precipitated form and is therefore not available at the start of the amplification reaction. After the temperature increased during the first thermal cycle formation, the precipitate dissolved and Mg2+ became fully available within the first 3 cycles. Such solutions have been shown to be fully applicable and capable of providing good hot start results. On the other hand, such solutions do not allow the preparation of mastermixes (mastermixes) containing all the reagents required for performing the nucleic acid amplification reaction except for the primers and the target nucleic acid. Thus, inter-assay data reproducibility and data comparison become complicated.

In view of the outlined prior art, it is an object of the present invention to provide improved alternative compositions and methods for hot start PCR, which allow to suppress non-specific priming and primer extension not only before the amplification process itself but also during the thermocycling process. More precisely, it is an object of the present invention to provide alternative compositions and methods for hot start PCR, wherein no extension of non-specifically annealed primers occurs.

Summary of The Invention

Accordingly, in a first aspect, the present invention provides a composition comprising

DNA polymerase

-deoxynucleotides

-at least one primer oligonucleotide, and

-a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety.

Such compositions are particularly useful for the performance of PCR amplification reactions because the formation of artificial amplification products, such as primer dimers, is avoided.

Preferably, the modification is located at the 5' end of the randomized oligonucleotide. Also preferably, said organic hydrophobic moiety of said modification is a pyrene or stilbene.

In a specific embodiment, the DNA polymerase is a thermostable DNA polymerase, such as Taq polymerase. If this is the case, the composition may comprise not only one primer but also at least one pair of amplification primers.

Furthermore, it is also within the scope of the present invention if any one of the compositions as defined above further comprises a target nucleic acid sample.

In a second aspect, the invention relates to a kit comprising a DNA polymerase and a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety. Preferably, the modification is located at the 5' end of the randomized oligonucleotide (capped random oligonucleotide). Also preferably, said organic hydrophobic moiety of said modification is a pyrene or stilbene.

In a specific embodiment, the kit is characterized in that the DNA polymerase is a thermostable DNA polymerase, such as taq DNA polymerase. If this is the case, the kit may comprise not only one primer but also at least one pair of amplification primers.

In a third aspect, the present invention provides a method for primer extension for a specific target nucleic acid, comprising the following steps

-providing a sample suspected of comprising said target nucleic acid

-adding any one of the compositions as disclosed above, and

-performing at least a first primer extension reaction.

Specifically, the present invention provides a method for amplifying a specific target nucleic acid, which comprises the following steps

-providing a sample suspected of comprising said target nucleic acid

-adding a composition as disclosed above comprising a thermostable DNA polymerase and a pair of amplification primers, and

-performing a nucleic acid amplification reaction.

Preferably, the nucleic acid amplification reaction is a polymerase chain reaction monitored in real time. In particular embodiments, melting curve analysis is then performed on the amplification product produced by the amplification.

Detailed Description

The present invention provides new and improved schemes for performing primer extension reactions with increased specificity. In particular, the present invention provides new and improved protocols for performing nucleic acid amplification reactions with increased specificity. The so-called hot start effect results in an effective inhibition of unwanted primer extension. Undesired primer extension results from a sporadic hybridization event, wherein a primer at least partially hybridizes to any sequence in the nucleic acid sample that is different from the actual primer binding side of the nucleic acid target.

The present invention therefore provides a composition comprising

DNA polymerase

-deoxynucleotides

-at least one primer oligonucleotide, and

-a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety.

The DNA polymerase in general may be any enzyme capable of performing a template-dependent primer extension reaction. Such template-dependent primer extension reactions can occur on all partially double-stranded nucleic acid hybrids characterized in that a primer nucleic acid having a free 3 'hydroxyl group hybridizes to a template nucleic acid having a single-stranded 5' overhang. The template-dependent polymerase then catalyzes extension of the 3' end of the primer by incorporating nucleotide residues that are consistently complementary to the nucleotides at opposite positions within the template strand. This reaction uses dNTPs as substrates and results in the release of pyrophosphate.

In one embodiment, the DNA polymerase is an RNA template-dependent polymerase or any modification thereof. Such enzymes are commonly referred to as reverse transcriptases. Examples are AMV reverse transcriptase or MMLV reverse transcriptase. In particular, Transcriptor reverse transcriptase (Roche applied science catalog No.: 03531317001) is an enzyme that can be used in the context of the present invention. The inventive compositions comprising such RNA-dependent DNA polymerases are particularly useful for the preparation and analysis of all kinds and applications of cDNA synthesis, and in particular 2-step RT-PCR.

In another embodiment, the DNA polymerase is a DNA template-dependent DNA polymerase or any mutant or modification thereof. A notable example is Klenow (Klenow) polymerase (Roche applied science catalog number 11008404001). Preferably, the DNA polymerase is a thermostable DNA polymerase or any mutant or modification thereof. A general example is TaqDNA polymerase from Thermus aquaticus (Thermusaquaticus) (Roche applied science catalog No.: 11647679001). The DNA-dependent DNA polymerase may or may not have 3 '-5' proofreading activity, for example Pwo polymerase (Roche applied science Cat: 11644947001). Furthermore, the DNA polymerase component of the invention may be a mixture of enzymes with and without proofreading activity, for example an extended high fidelity system (ExpandhFidelitysys) (Roche applied science catalog No.: 11732641001). The inventive compositions comprising any kind of thermostable polymerase are particularly useful for performing various preparative or analytical embodiments of the Polymerase Chain Reaction (PCR).

In a further embodiment, the DNA polymerase component of the invention is a thermostable DNA-dependent DNA polymerase with additional RNA template-dependent reverse transcriptase activity, such as a polymerase from Thermus thermophilus (Thermusthermophilus) (RocheApplied science catalog No.: 11480014001), or a mixture of an RNA-dependent DNA polymerase (i.e., a reverse transcriptase) and a thermostable DNA-dependent DNA polymerase. The inventive compositions comprising such components are particularly useful for analytical performance of one-step RT-PCR.

Deoxynucleotide (deocxynuclotide) triphosphates (dNTPs) are typically a mixture of dATP, dCTP, dGTP and dTTP, however, in some particular cases, only 3 or fewer different kinds of dNTPs may be used. Furthermore, such dntps may be chemically modified in any manner so long as the building block is still capable of being incorporated into the nascent polynucleotide strand by the polymerase. For example, the modified nucleotide compounds may carry biotin or fluorescent compound modifications at the respective base moiety.

The at least one primer oligonucleotide is typically a deoxyoligonucleotide that is completely or almost completely complementary to a specific region of the target nucleic acid. Furthermore, the primer moiety must have a free 3' hydroxyl group so that it can be extended by a DNA polymerase. For specific purposes, such primers may be chemically modified, for example, at their 3' end. Examples of commonly used modifications are biotin labeling, digoxigenin (digoxgenin) labeling and fluorescent labeling.

If a thermostable DNA-dependent DNA polymerase is designed for a PCR reaction, the composition according to the invention typically comprises 2 primer oligonucleotides that hybridize in opposite directions to opposite strands of the target nucleic acid adjacent to the target sequence to be amplified. It is also possible that the composition of the invention comprises multiple pairs of oligonucleotide PCR primers for multiplex PCR amplification.

An important compound to distinguish the composition according to the invention from compositions currently used in the art is the addition of randomized 5-8 mer oligonucleotides, characterized in that said oligonucleotides comprise a modification with an organic hydrophobic moiety. More precisely, the term "randomized oligonucleotide" refers to a pool of oligonucleotides whose sequence represents all possible combinations of more or less equal 4 different nucleotide residues. Although the addition of 5-mers as well as the addition of 8-mers has proven to have the desired hot start effect, it has proven to be particularly advantageous if randomized hexamer oligonucleotides are used. The randomized oligonucleotide may be added to a primer extension reaction or PCR reaction at a concentration ranging from 10. mu.M to 1mM, preferably 25. mu.M to 400. mu.M, and most preferably at a concentration of about 100. mu.M. It has also proved to be particularly advantageous if the randomized oligonucleotide has a non-extendable 3' end, which can be blocked, for example, by a phosphate moiety. This avoids unwanted extension by the polymerase in case of accidental hybridization of any oligonucleotide at any region of the sample nucleic acid.

Randomized oligonucleotides are chemically modified with an organic hydrophobic moiety. The moiety does not generally interfere with any type of primer extension reaction. For example, such organic hydrophobic moieties may be selected from the group consisting of condensed (condensed) aromatic and heteroaromatic rings, such as naphthalene (naphthalin), anthracene (anthracen), phenanthrene (phenantren), pyrene, anthraquinone, carbazole phenanthroline (carbalphenylantrolines), quinoline (quonolines), etc., or from stilbene (stilbens), or from steroids such as cholesterol. Such hydrophobic moieties may be substituted with bulky substituents such as cyano, methoxy, methyl, nitro and halogen, and moieties are known to act as so-called "caps" for stabilizing terminal base pairs. Narayanan, S., et al, nucleic acids research32(9) (2004) 2901-2911; dogan, Z, et al, journal of American chemical society126(15) (2004) 4762-4763.

Most preferably, such organic hydrophobic moiety is an optionally substituted pyrene or an optionally substituted stilbene (Stilben) having the following chemical structure:

most preferably, such pyrene or stilbene is attached to the 5 'end of a randomized oligonucleotide, and the 5' end of such oligonucleotide has the following structure:

the organic hydrophobic moiety may be located at any portion of the randomized oligonucleotide. Preferably, however, the modification is introduced at the 5' end of the randomized oligonucleotide. The reason is that such 5' modifications can be introduced into the oligonucleotide using phosphoramidite chemistry with appropriate terminal phosphoramidites according to standard methods well known in the art, and pyrene and stilbene phosphoramidites are commercially available.

The randomized oligonucleotide may comprise nucleobase (nucleobase) analogs with modified bases such as 7 deaza analogs such as 7 deaza dG, 7 deaza 8 aza analogs such as 7 bromo 7 deaza 8 aza 2 amino dA, or substituted bases such as propynyl U, propynyl C, or analogs with modified sugars such as 2' methoxy ribose or locked sugars such as in LNA, or with ribose analogs such as hexitol and altritol. Instead of randomization universal bases such as nitroindole or N8 ribosylation-7 deaza 8 aza dA are used, whereas it is preferred to use universal bases instead of random oligonucleotides at only one position of the random oligonucleotides. The internucleoside phosphate may be substituted by a phosphate mimic (mimetikum), such as a phosphorothioate or methylphosphonate or a phosphoramidate. The randomized oligonucleotides preferably have one hydrophobic moiety but may be additionally substituted by other hydrophobic moieties, while the hydrophobic moieties are independently selected from each other.

Those compositions comprising randomized oligonucleotides chemically modified with organic hydrophobic moieties along with a DNA-dependent thermostable DNA polymerase and at least one pair of amplification primers are particularly useful for performing PCR amplification reactions. The reason is that the presence of the randomized and modified oligonucleotides effectively inhibits the formation of artificial amplification products, such as primer dimers, at temperatures below the annealing temperature of the respective amplification primers, thereby generating a hot start effect.

It is also within the scope of the present invention if any of the compositions as defined above further comprises a target nucleic acid sample. The sample may typically comprise, for example, genomic DNA or fragmented genomic DNA together with a DNA-dependent DNA polymerase, or total cellular or poly a + RNA together with an RNA-dependent DNA polymerase.

In a particular aspect, the invention also provides a kit for preparing a composition as disclosed in detail above. Thus, the invention also relates to a kit comprising at least a DNA polymerase and a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety. Preferably, the modification is any of the examples as disclosed above and is located at the 5' end of the randomized oligonucleotide. In addition, the kit may comprise further components, such as deoxynucleotide triphosphates (dNTPs) and suitable buffers and other reagent additives, which are used to perform the respective primer extension reactions. In addition, the parameter specific kit may comprise at least one target-specific primer oligonucleotide.

In a first embodiment, the kit is designed for cDNA synthesis and comprises a reverse transcriptase as disclosed above. As a primer component, the kit may contain any parameter-specific primer for amplifying specific cDNAs.

In a second embodiment, the kit is designed for performing PCR and comprises a DNA-dependent thermostable polymerase or a mixture of DNA-dependent thermostable polymerases. The kit may then additionally comprise, for example, dNTPs and/or buffer solutions and/or at least one or more pairs of amplification primers. More specifically, if the kit is designed for one-step RT-PCR, the enzyme component may be a DNA-dependent thermostable DNA polymerase, which additionally comprises reverse transcriptase activity.

In a third particular embodiment, the kit is designed for 2-step RT-PCR and may comprise various combinations of components selected from the components of the first and second embodiments as disclosed above.

Furthermore, the kit according to the second and third embodiments may comprise components for detecting PCR amplification products. For example, if the kit is designed for real-time PCR (qPCR), such a kit may additionally comprise a double stranded DNA binding dye component, such as SybrGreen (Roche applied science catalog # 04707516001) or LC480ResoLight dye (Roche applied science catalog # 04909640001). Alternatively, such a kit may additionally comprise fluorescently labeled hybridization probes, such as TaqMan probes (U.S. Pat. No. 5,804,375), molecular beacons (U.S. Pat. No. 5,118,801), FRET hybridization probes (U.S. Pat. No. 6,174,670) or simple probes (SimpleProbes) (WO 02/14555).

The present invention relates not only to compositions and kits, but also to methods of performing primer extension reactions in general and PCR or reverse transcription reactions in particular. Thus, in its broadest sense, the method according to the invention comprises the following steps

-providing a sample suspected of comprising said target nucleic acid

-adding any one of the compositions as disclosed above, and

-performing at least a first primer extension reaction.

More precisely, the method according to the invention comprises the following steps

-providing a sample suspected of comprising said target nucleic acid

-addition of

DNA polymerase

-deoxynucleotides

-at least one primer oligonucleotide, and

-a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety,

-performing at least a first primer extension reaction.

In a first embodiment, the sample is total or poly a + RNA, the DNA polymerase is a reverse transcriptase, and the primer oligonucleotide is a specific primer complementary to a specific type of cDNA.

In a second embodiment, the sample is derived from genomic DNA, the DNA polymerase is a thermostable DNA polymerase or a mixture of thermostable DNA polymerases, and the at least one or more pairs of amplification primers are added prior to the PCR amplification reaction. Preferably, the nucleic acid amplification reaction is a polymerase chain reaction monitored in real time according to standard methods known in the art (see, e.g., US5,210,015, US5,338,848, US5,487,972, WO97/46707, WO97/46712, WO 97/46714).

In particular embodiments, melting curve analysis is performed on the resulting amplification products by subjecting the amplification products to a thermal gradient over time (US6,174,670, US6,569,627). In this type of experiment, the fluorescence intensity due to the binding of the respectively labeled hybridization probes or due to fluorescence originating from the DNA binding dye is monitored. Subsequently, the first derivative of the decrease in fluorescence intensity due to melting of both strands of the hybridization probe or amplicon, respectively, is plotted against the temperature gradient. As will be shown in the examples, the presence of randomized 5-8 mer oligonucleotides during the amplification process subsequently provides good melting curve results, characterized in that the oligonucleotides comprise a modification with an organic hydrophobic moiety.

In summary, it can be stated that the method of the invention comprises several advantages over the methods already disclosed in the art. The presence of randomized 5-8 mer oligonucleotides during primer extension reactions such as reverse transcription or PCR or RT-PCR apparently leads to an increase in the specificity of the respective reaction, characterized in that said oligonucleotides comprise a modification with an organic hydrophobic moiety.

To date, the inventors have not fully appreciated one or more reasons for such positive effects. One possible mechanistic explanation may be that at low temperatures, part of the randomized population of oligonucleotide molecules may interact with the primer to the extent that the primer cannot be extended, even though it has annealed to a longer, substantially complementary nucleic acid molecule.

One of the main advantages of the present invention is the ease of use and short activation time to eliminate inhibition of the polymerase at low temperatures. Briefly, randomized 5-8 mer oligonucleotides are added to a PCR reaction set-up, characterized in that the oligonucleotides comprise modifications with organic hydrophobic moieties. During PCR thermal cycling, the denaturation time prior to the first cycle typically required to separate a double-stranded DNA template into single strands is sufficient to eliminate the interaction between the conjugated random oligonucleotides and the PCR primers.

In addition, random hexamers can be synthesized according to standard phosphoramidate chemistry well established in the art. Furthermore, also 5' modifications can be very easily introduced into the oligonucleotide using phosphoramidate chemistry with a separately modified terminal phosphoramidate according to standard methods. Thus, the production costs for the PCR additive according to the invention are considerably lower compared to other hot start solutions.

Furthermore, the methods, compositions and kits of the invention can be used for any kind of primer extension, reverse transcription or PCR amplification in general, regardless of which specific target nucleic acid sequence should be prepared, amplified, detected or analyzed.

Brief Description of Drawings

FIG. 1 amplification of genomic DNA in the Presence of pyrene-capped hexamers according to example 1

Lane 1: additive-free PCR, 50ng DNA

Lane 2: PCR, 25ng DNA without additive

Lane 3: PCR, 10ng DNA without additive

Lane 4: additive-free PCR, 5ng DNA

Lane 5: additive-free PCR, 1ng DNA

Lane 6: PCR without additives, no template control

Lane 7: additive-containing PCR, 50ng DNA

Lane 8: PCR, 25ng DNA with additive

Lane 9: PCR, 10ng DNA with additive

Lane 10: PCR, 5ng DNA containing additive

Lane 11: PCR, 1ng DNA with additive

Lane 12: PCR with additives, no template control

FIG. 2 amplification of genomic DNA in the Presence of pyrene-capped hexamers according to example 2

Lane 1: taq polymerase, 30ng DNA

Lane 2: taq polymerase, 3ng DNA

Lane 3: taq polymerase, 0.3ng DNA

Lane 4: taq polymerase, pyrene-capped hexamer, 30ng DNA

Lane 5: taq polymerase, pyrene-capped hexamer, 3ng DNA

Lane 6: taq polymerase, pyrene-capped hexamer, 0.3ng DNA

FIG. 3 amplification according to example 4 in the Presence of pyrene-or stilbene-capped octamers

Lanes 1-6: PCR products formed with 50ng, 25ng, 10ng, 5ng, 1ng and 0ng human DNA, respectively, in the absence of additives.

Lanes 7-12: PCR products formed with 50ng, 25ng, 10ng, 5ng, 1ng and 0ng of human DNA, respectively, in the presence of 100M pyrene-capped octamer.

Lanes 13-18: PCR products formed with 50ng, 25ng, 10ng, 5ng, 1ng and 0ng of human DNA, respectively, in the presence of 100M stilbene-capped octamer.

FIG. 4 real-time PCR melting Curve analysis according to example 7

FIG. 4a melting curve analysis for PCR and pyrene-free capped hexamer free of various amounts of genomic DNA

FIG. 4b PCR and melting curve analysis of pyrene-capped hexamers with various amounts of genomic DNA

FIG. 5 real-time RTPCR according to example 9

5 a: first strand cDNA synthesis in the presence of a capped hexamer, and subsequent PCR in the absence of a capped hexamer,

5 b: first strand cDNA synthesis in the presence of a capped hexamer, and subsequent PCR in the presence of a capped hexamer,

5 c: first strand cDNA synthesis in the absence of capped hexamers, and subsequent PCR in the absence of capped hexamers,

5 d: first strand cDNA synthesis in the absence of capped hexamers, and subsequent PCR in the presence of capped hexamers.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made in the procedures set forth without departing from the spirit of the invention.

Examples

Randomized oligonucleotides were synthesized in the trityl off (trityloff) mode on a 10 μmol scale on an ABI394 synthesizer by standard methods using commercially available CPG phosphate (2- [2- (4, 4' -dimethoxytrityloxy) ethanesulfonyl ] ethyl-2-succinyl (succinoyl) -long-chain alkylamino-CPG) as a solid support and an equimolar (∑ 0.1mol) mixture of standard da (bz) dT, dg (ibu) dc (bz) phosphoramidites, deprotected with ammonia or NaOH under standard conditions and desalted via dialysis to the product.

Example 1

5' pyrene-capped hexamers were analyzed in DNA amplification. PCR reactions in the presence or absence of 100. mu.M pyrene-capped hexamers were performed in 50. mu.l reactions containing 50ng, 25ng, 10ng, 5ng, 1ng and 0ng human genomic DNA, 30mM Tris-HCl, pH8.6, 1.5mM MgCl₂50mM KCl, 0.2mM of each dNTP's, 0.4. mu.M primer (SEQ ID NO: 1ATTAGAGAACCATGTTAACACTACCG and SEQ ID NO: 2GAGGTGAATGACCACTGTTTATTTTC) and 2.5 units Taq DNA polymerase. The following cycling conditions were used: initial denaturation at 94 ℃ for 4 minutes, and 35 cycles of denaturation at 94 ℃ for 20 seconds, annealing at 62 ℃ for 30 seconds, extension at 72 ℃ for 60 seconds, and a final extension step at 72 ℃ for 7 minutes. The amplification products were separated on an agarose gel and visualized by ethidium bromide staining.

The results depicted in FIG. 1 show a clear improvement in amplification specificity in the presence of pyrene-capped hexamers.

Example 2

5' pyrene-capped hexamers were analyzed in real-time PCR. PCR reactions in the presence or absence of pyrene-capped hexamers were performed in 20. mu.l reactions containing 30ng, 3ng or 0.3ng human genomic DNA, 50mM Tris-HCl, pH8.6, 0.2mM CHAPS, 1mM Guigcap, 20mM KCl, 3mM MgCl₂0.4. mu.M primer (SEQ ID NO: 3GGAAGTACAGCTCAGAGTTCTGC and SEQ ID NO: 4GAATCTCCATTCATTCTCAAAAGGACT), 0.2mM deoxynucleotide and 2.5 units TaqDNA polymerase. PCR inThe following cycling conditions were used in the 480 apparatus: initial denaturation at 95 ℃ for 2 minutes, and 45 cycles of denaturation at 95 ℃ for 1 second, annealing at 65 ℃ for 10 seconds, and extension at 72 ℃ for 10 seconds. The amplification products were separated on an agarose gel and visualized by ethidium bromide staining (FIG. 2). Result display deviceA clear improvement in amplification specificity for the pyrene-capped hexamer.

Example 3

The 5' pyrene-capped pentamer was analyzed in the same experimental setup as described in example 2. The final concentrations tested were 50. mu.M, 100. mu.M, 150. mu.M and 200. mu.M. In the control reaction (no additive present) multiple PCR products were formed, whereas in the presence of increasing amounts of pyrene-capped pentamers the desired product was formed with increased yield. Specificity and sensitivity were significantly higher than control experiments without additives (not shown).

Example 4

The 5' pyrene-capped octamer and stilbene-capped octamer were tested at a final concentration of 100. mu.M in the amplification of human collagen gene fragments using 50ng, 25ng, 10ng, 5ng, 1ng and 0ng of human DNA, using the same PCR buffer as described in example 1. PCR primers (SEQ ID NO: 5TAAAGGGTCACCGTGGCTTC and SEQ ID NO: 6CGAACCACATTGGCATCATC) were used at a concentration of 0.4. mu.M. The total reaction volume was 50. mu.l. The PCR cycles were performed in a block cycler (blockcycler) which used an initial denaturation at 94 ℃ for 4 minutes, and 35 cycles at 94 ℃ for 20 seconds, 62 ℃ for 30 seconds, 72 ℃ for 4 minutes, and a final extension step at 72 ℃ for 7 minutes.

The results are depicted in fig. 3. In the absence of additives, a nonspecific product of about 550bp was formed. No non-specific products were observed in the presence of the capped oligonucleotide. Stilbene-capped octamers lead to strong fluorescence at the bottom of the gel.

Example 5

5' pyrene-capped monomers were analyzed in real-time PCR. In the presence or absence of pyrene-capped monomers (up to 400. mu.M) or pyrene-capped hexamers (up to 400. mu.M)The PCR reaction was carried out in a 20. mu.l reaction containing 30ng, 3ng, 0.3ng, 0.03ng, 0.01ng and 0ng of human genomic DNA, 50mM Tris-HCl, pH8.6, 0.2mM CHAPS, 1mM BCHP, 20mM KCl, 3mM MgCl₂0.4. mu.M primer (SEQ ID NO: 7CACCCCGTGCTGCTGACCGA and SEQ ID NO: 8AGGGAGGCGGCCACCAGAAG), 0.2mM deoxynucleotide and 2.5 units TaqDNA polymerase. PCR inThe following cycling conditions were used in the 480 apparatus: initial denaturation at 95 ℃ for 2 minutes, and 45 cycles of denaturation at 95 ℃ for 1 second, annealing at 65 ℃ for 15 seconds, and extension at 72 ℃ for 5 seconds. The amplification products were separated on an agarose gel and visualized by ethidium bromide staining. The results show a clear improvement in amplification specificity by pyrene-capped hexamers compared to the control reaction (not shown), but no increase in specificity with pyrene-capped monomers.

Example 6

The 3 'phosphorylated hexamers without organic molecules at the 5' terminus were tested in the amplification of human collagen gene fragments at final concentrations up to 200 μ M using 50ng, 25ng, 10ng, 5ng, 1ng and 0ng of human genomic DNA, using the same PCR buffer as described in example 1. PCR primers (SEQ ID NO: 5TAAAGGGTCACCGTGGCTTC and SEQ ID NO: 6CGAACCACATTGGCATCATC) were used at a concentration of 0.4. mu.M. The total reaction volume was 50. mu.l. PCR cycles were performed in a block cycler using an initial denaturation step at 94 ℃ for 4 minutes, 35 cycles at 94 ℃ for 20 seconds, 58 ℃ for 30 seconds, 72 ℃ for 4 minutes, and a final extension step at 72 ℃ for 7 minutes. The amplification products were separated on an agarose gel and visualized by ethidium bromide staining. The results showed no difference in PCR product composition, regardless of the presence or absence of hexamers (not shown).

Example 7

5' pyrene capped hexamerThe body was analyzed in real-time PCR. PCR reactions in the presence or absence of pyrene-capped hexamers were performed in 20. mu.l reactions containing 30ng, 3ng, or 0.3ng, 0.03ng, 0.01ng, and 0ng human genomic DNA, 50mM Tris-HCl, pH8.6, 0.2mM HAPS, 1mM Guigcap, 20mM KCl, 3mM MgCl₂0.4. mu.M primer (SEQ ID NO: 3GGAAGTACAGCTCAGAGTTCTGC and SEQ ID NO: 4GAATCTCCATTCATTCTCAAAAGGACT), 0.2mM deoxynucleotides and 2.5 units of TaqDNA polymerase and SYBRGreen (1: 40000). PCR inThe following cycling conditions were used in the 480 apparatus: initial denaturation at 95 ℃ for 2 minutes, and 45 cycles of denaturation at 95 ℃ for 1 second, annealing at 65 ℃ for 10 seconds, and extension at 72 ℃ for 10 seconds. For relative quantification of specific and non-specific products, melting curves were performed according to the protocol recommended for LightCycler 480. The results are shown in fig. 4. In the absence of additive (fig. 4A), a large amount of non-specific product was formed, and in the presence of additive (fig. 4B), the non-specific product was significantly reduced.

Example 8

To test whether the 5' pyrene-capped hexamer could also be used in RT-PCR using another DNA polymerase and whether the additives affected the cp value of the PCR reaction. We therefore used a Tth polymerase based480RNA Master hydrolysis probes (MasterHydrolysisProbes) (Roche applied science, Cat. No. 04991885001) were subjected to RT-PCR reaction. Mu.l of the reaction mixture contained 100pg, 10pg, 1pg, 0.1pg and 0pg, respectively, of total RNA from human hepatocytes, 7.4. mu.l of RNAMaster, 3.25mM manganese acetate, 0.5. mu.M of each primer (SEQ ID NO: 9TGCAGCCTCCATAACCATGAG and SEQ ID NO: 10GATGCCTGCCATTGGACCTA) and 0.25. mu.M of hydrolysis probe (SEQ ID NO: 11 FAM-GATGCCTGCCATTGGACCTA-TAMRA). Inversion in the absence of pyrene-capped hexamers or with 50. mu.M, 100. mu.M or 200. mu.M pyrene-capped hexamersShould be used. In thatThe RT-PCR was performed in 480 instruments according to the protocol recommended by the manufacturer. The cp values are shown in table 1. Pyrene-capped hexamers had no effect on the crossover point (crossingpoint). There is no delay or loss of sensitivity in the amplified signal.

Table 1:

example 9

To assess whether an increase in sensitivity could also be observed in cDNA synthesis, we performed two-step RT-PCR experiments in a reaction setup close to the conditions of one-step RT-PCR. 4 reactions were performed in parallel:

(a) first strand cDNA synthesis without capped random hexamers, and subsequent PCR in the presence of capped random hexamers,

(b) first strand cDNA synthesis without 5' capped random hexamers, and subsequent PCR in the absence of capped random hexamers,

(c) first strand cDNA synthesis in the presence of 5' capped random hexamers, and subsequent PCR in the absence of capped random hexamers,

(d) first strand cDNA synthesis in the presence of 5' capped random hexamers, and subsequent PCR in the presence of capped random hexamers.

Selecting primer pairs that cause non-specific product formation when a small amount of RNA is present in the RT-PCR reaction: the G6PDHForw (SEQ ID NO: 12GCAAACAGAGTGAGCCCTTC) and G6PDHrev (SEQ ID NO: 13GGGCAAAGAAGTCCTCCAG) primers. cDNA was synthesized in a 20. mu.l reaction containing 0.5. mu.M primer, 0.6 monoBit Transcriptor (Roche applied sciences, Cat. No.: 03531317001), 30mM Tris. HCl, pH 8.6; 3mM MgCl₂200. mu.M dATP, 200. mu.M dGTP, 200. mu.M dCTP, 600. mu.M dUTP, 20M KCl, 0.2M CHAPSO, 1M Grigcap, 125ng/mlT4gene32 protein, SYBRGreen at a final dilution of 1: 20000 and 10pg total RNA from HeLa cells. 2 samples were prepared for cDNA synthesis, one containing 100. mu.M pyrene-capped hexamer and the other containing no pyrene-capped hexamer. The reaction was incubated at 50 ℃ for 10 minutes, at 95 ℃ for 2 minutes and cooled on ice. PCR was performed in a 20. mu.l reaction volume using 2. mu.l of the DNA reaction mix, 0.5. mu.M primer, 1.2 units of Taq polymerase in the same buffer as described for the cDNA reaction mix, in the presence or absence of a final concentration of 100. mu.M of additional pyrene-capped hexamer. The reaction was incubated in a LightCycler480 instrument at 95 ℃ for 2 minutes and 45 cycles of 95 ℃/10 seconds, 60 ℃/10 seconds, 72 ℃/13 seconds. Melting profiles of the amplification products are shown in FIGS. 5 a-d. In reactions where pyrene-capped hexamers were present during cDNA synthesis (5a and 5b), one single product with the expected melting temperature was formed. When cDNA synthesis was performed in the absence of pyrene-capped hexamers (5c and 5d), several products were produced with melting temperatures different from that of the specific product. This result shows that pyrene-capped hexamers can inhibit non-specific product formation during reverse transcription of RNA.

Example 10

Parallel analysis was performed in DNA amplification with 100. mu.M 5 ' pyrene-capped hexamers (pyrene-CGGTAG-3 ' phosphate) overlapping 6 base pairs at the 3 ' end of one of the primers and 100. mu.M 5 ' pyrene-capped hexamers (pyrene-CTTTTA-3 ' phosphate) not complementary to the primers. In a 50. mu.l reaction, it contained 50ng, 25ng, 10ng, 5ng, 1ng and 0ng of human genomic DNA, 30mM Tris-HCl, pH8.6, 1.5mM MgCl₂50mM KCl, 0.2mM of each dNTP's, 0.4. mu.M primer (SEQ ID NO: 1ATTAGAGAACCATGTTAACACTACCG and SEQ ID NO: 2GAGGTGAATGACCACTGTTTATTTTC) and 2.5 units Taq DNA polymerase. PCR was carried out in a block cycler under the following cycling conditionsLine: initial denaturation at 94 ℃ for 4 minutes, and 35 cycles of denaturation at 94 ℃ for 20 seconds, annealing at 62 ℃ for 30 seconds, extension at 72 ℃ for 60 seconds, and a final extension step at 72 ℃ for 7 minutes. The amplification products were separated on an agarose gel and visualized by ethidium bromide staining. The control reactions in the absence of additives were run in parallel. The PCR product could not be detected in the presence of pyrene-CGGTAG-3' phosphate. In the samples containing non-complementary pyrene-CTTTTA-phosphate, similar product yield and sensitivity (in the absence of any additives) as in the control reaction were achieved.

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Claims (9)

1. A composition comprising

DNA polymerase

-deoxynucleotides

At least one primer oligonucleotide

-a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety, wherein said modification is located at the 5' end of said randomized oligonucleotide,

and wherein the randomized oligonucleotide has a non-extendable 3' end, and wherein the moiety is pyrene or stilbene.

2. Composition according to claim 1, characterized in that

-the DNA polymerase is thermostable, and

-said composition comprises a pair of amplification primers.

3. The composition of claim 1, further comprising a target nucleic acid sample.

4. A kit comprising

A DNA polymerase, and

-a randomized 5-8 mer oligonucleotide, characterized in that said oligonucleotide comprises a modification with an organic hydrophobic moiety, wherein said modification is located at the 5 'end of said randomized oligonucleotide, and wherein said randomized oligonucleotide has a non extendable 3' end, and wherein said moiety is pyrene or stilbene.

5. The kit according to claim 4, characterized in that the DNA polymerase is thermostable, further comprising a pair of amplification primers.

6. Method for amplifying a specific target nucleic acid comprising the following steps

-providing a sample suspected of comprising said target nucleic acid

-adding a composition according to claims 1-2

-performing at least a first primer extension reaction.

7. The method according to claim 6, wherein the composition is a composition according to claim 2, further comprising the step of performing a nucleic acid amplification reaction.

8. The method according to claim 7, wherein said nucleic acid amplification reaction is a real-time monitored polymerase chain reaction.

9. Method according to claims 5-6, characterized in that the amplification products produced by the amplification are subjected to a melting curve analysis.