DE102004009704A1 - New conjugates useful for labeling nucleic acids comprise a label coupled to nucleotide or nucleoside molecules through polymer linkers - Google Patents

Abstract

Conjugates (I) comprising a label coupled to 1-100 nucleotide or nucleoside molecules through polymer linkers with a length of 100-20000 atoms are new. Conjugates of formula (I) are new. (Nuc-Linker)n-Label (I) Nuc : a nucleotide or nucleoside; Linker : a water-soluble polymer chain with a length of 100-20000 atoms; Label : a label; n : 1-100. Independent claims are also included for: (1) oligonucleotides or polynucleotides including at least one molecule of (I) per nucleic acid chain; (2) modifying nucleic acid chains using (I) for coupling.

Description

1. Introduction

1.1 classification in the technological field

The The present invention relates to the manufacture and use modified nucleotide and nucleoside building blocks, hereinafter "nuc macromolecules." Oligo- or polynucleotides are only subject of the invention when used the nuc-macromolecules according to the invention have been produced.

1.2 State of the art:

nucleotides and nucleosides play a central role in different Metabolic processes of living organisms ("biochemistry and Pathobiochemistry ", G. Löffler, 2003) and are common building blocks in modern biotechnology ("Molecular Cloning", J. Sambrook, vol 1-3, 2001, ISBN 0-87969-576-5), for example for artificial detection systems ("DNA Microarrays", Bowtell, 2003, ISBN 0-87969-624-9, "Microarray biochip Technology "M Schena, 2000, ISBN 1-881299-37-6). For this reason, modified nucleotides, nucleotide analogs, in different areas of biotechnology, medicine and pharmacology used ("Nucleotide Analogous, "Scheit, 1980, ISBN 0-471-04854-2, "Nucleosides and Nucleic Acid Chemistry ", Kısakürek 2000, "Anti-HIV Nucleosides "Mitsuya, 1997, "Nucleosides Analogs in cancer therapy ", Cheson, 1997).

One The most important fields of modern life science are analyzes of Nucleic acids. A big area This analysis deals with the detection of nucleic acids or their components in biological preparation. In many cases will For this purpose, a reactant labeled with the to be analyzed nucleic acid can react. The person skilled in the art has different possibilities for labeling the nucleic acids known. On the one hand can individual nucleic acid components, On the other hand, short nucleotides or nucleosides may be modified Fragments of nucleic acids, Oligonucleotides or polynucleotides are used for detection.

Conventional modified nucleotides are described, for example, in Lee et al. Nucleic acid research 1992, V. 20, p.2471; Augustin et. al. J. Biotechnology 2001, V. 86, pages 289-301; U.S. Patent 4,828,979; Held et al. Nucleic acid research, 2002, v. 30 P. 3857. These nucleotides have a low molecular weight detectable part directly (such as a fluorescent dye molecule) or can be indirectly detected (such as in a biotin molecule, the first detected after coupling with a streptavidin-dye conjugate becomes)). Such nucleotides are examples of the state of the art Nucleotide modifications. Many modified nucleotides can be commercial, be purchased, e.g. by NEN Life Science Products (Biotin-11-dUTP, DNP-11-dATP, fluorescein-12-dCTP), from Amersham Bioscience dCTP-Cy3, dCTP-Cy5) or from Roche (Biotin-16-dUTP). Appropriate detection reagents, e.g. labeled streptavidin, labeled antibodies, may be from the same suppliers be obtained. Also modified nucleosides are used for detection of nucleic acids used (Trilink Biotechnologies, Eurogentec, MWG-Biotech).

to Simplification of the presentation will be modified nucleotides below discussed. It should be obvious to a person skilled in the art that also modified nucleosides for the enzymatic and non-enzymatic syntheses are used can, Conventionally modified nucleosides are available, for example, from Trilink Biotechnologies or Eurogentec.

Conventional modified nucleotides have an equimolar ratio between the nucleotide component and the low molecular detectable Part, e.g. Dye molecule or biotin molecule. Between the two Sharing is a linker with an average length of 5 - 30 Atoms.

Such nucleotides can be incorporated by polymerases into the growing strand and thus introduce a signal-carrying molecule (dye) or signal-transmitting molecule (biotin or digoxigenin) into the nucleic acid chain. Following incorporation of modified nucleotides, signal detection may be direct in the case of dye-labeled NT, or after incubation with a secondary signal-carrying molecule (eg, strepavidin-dye conjugate in the case of biotin) when signal-transmitting molecules are used. The subsequent coupling of the signal-carrying molecules, such as streptavidin, often occurs with insufficient Yields of 20-60%.

Very often in the labeling of nucleic acids signal amplification steps used. these can be carried out at different stages of the analysis. Examples of this make material duplication (PCR), multiple incorporation of labeled nucleotides, multistep subsequent Labeling of biotin nucleotides ("Molecular Cloning", J. Sambrook, vol. 1-3, 2001, ISBN 0-87969-576-5). These methods lead to a distortion of the Signals, since they consist of several, often insufficiently controlled steps, which occur with different yields and many factors can be influenced.

The desired and for obvious to the person skilled signal amplification by a multiple Labeling of a nucleotide during the nucleotide synthesis proves in combination with conventional nucleotide structures as a very big one Disadvantage. Although you can such nucleotides produced without much effort (Example 25), but lose their function as a substrate for polymerases (Example 34B) and other enzymes. The cause lies in change the properties of the nucleotides as a result of coupling to a significant bigger molecule.

Certain Methods described, for example, in Seeger WO 0018956, Kartalov WO02072892, rely on the detection of signals from single nucleotide molecules. When using conventional nucleotides affect several phenomena such as e.g. bleaching the dyes or blinking the results the single molecule detection. For such Method means an increase in signal strength and intensity of nucleotide labeling a reduction in the error rate during detection.

It There is thus a need for modified nucleotides or nucleosides with an improved signaling or signal-transmitting effect, especially for their use for labeling nucleic acids in nucleic acid analysis.

A The object of the present invention is therefore the provision of modified nucleotides or nucleosides that have their substrate properties for polymerases or other enzymes, and after their incorporation into nucleic acids improved signal intensity to ensure their analysis.

Especially It is an object of the present invention to provide modified nucleotides or to provide nucleosides with more than one label can be coupled, without losing their substrate properties for polymerases or lose other enzymes.

A Another object of the present invention is the provision modified nucleotides or nucleosides, especially for use for labeling nucleic acids in nucleic acid analysis, ensure an improved signal-to-noise ratio, for example in the concentration ratio is increased from nucleotide to marker to be incorporated.

A Another object of the invention is the provision of the method for labeling nucleic acids with modified according to the invention Nucleotides.

These and further objects of the present invention are achieved by the Provision of the specified in the claims objects solved.

preferred embodiments are in the subclaims Are defined.

The The present invention overcomes the disadvantages of the prior art and describes a new class of modified nucleotides that Nuc-macromolecules. Nuc-macromolecules are characterized in that a or more nuc-components with one or more signaling or signal-transmitting macromolecular components (markers) are connected. The respective Nuk component preserved in a nuc macromolecule its substrate function and may e.g. by polymerases in one growing strand to be installed. A signaling macromolecular Component carries for example, several dye molecules.

Nuc-macromolecules may be used as conventionally modified nucleotides in various fields of biotechnology and medicine. In the following, only their use for the nucleic acid labeling should be shown as an example. Other uses of nuc macromolecules are the art man already known like that

1.3 Terms / Definitions:

1.3.1. Macromolecular compound - a Molecule, a molecular complex, a nanocrystal or nanoparticle whose mass is between 2kDa and 100kDa, 100kDa and 200kDa, 200kDa and 1000kDa or 1MDa and 100MDa or 100MDa and 100GDa. Examples of macromolecular compounds are nucleic acids, like Oligonucleotides of a length of more than 10 nucleotides, polynucleotides, polypeptides, proteins or enzymes, quantum dots, polymers such as PEG, Mowiol, polyacrylate, Nano gold particles.

1.3.2. Low molecular compound - a molecule or a molecular complex, whose mass is less than 2000 Da (2kDa), e.g. natural nucleotides, dATP, dUTP, many dyes such as Cy3, rhodamine, fluorescein, linker with an average length between 5 and 30 atoms, rare earths and conventionally modified Nucleotides, such as biotin-16-dUTP.

1.3.3. A nuc macromolecule in the context of this application is a chemical structure which has one or more nuc-components, one or more linker components and a marker component, 1 or 2 : (Nuk linker) _n marker in which:
Nuk - a nuk component is
Linker - is a linker component
Marker - is a Makrer component
n - is an integer between 1 and 100

In a preferred embodiment, the linker component consists of a coupling unit L for the coupling of the linker to the nuc-component, of a water-soluble polymer and of a coupling unit T for the coupling of the linker to the marker component. In this preferred embodiment, a nuc-macromolecule has the following structure, 1 or 2 : (Nuk-L polymer T) _n label in which:
Nuk - a nucleotide or a nucleoside is (Nuk component)
L - is a part of the linker that represents the link between Nuk and the linker residue (coupling unit L)
T - part of the linker is the link between the linker residue and the
Marker represents (coupling unit T)
Polymer - is a part of the linker which is a water-soluble polymer with an average length between 100 and 10,000 atoms (coupling unit L, polymer linker, coupling unit T are combined in this embodiment as a linker component)
Marker - a marker component
n - an integer between 1 and 100

Nuc-macromolecules are by a combination of one or more nuc-components, accordingly one or more long linker components and a marker component Are defined.

1.3.3.1 Nuc component

Nuk component can be both a nucleotide and a nucleoside. In the following, nucleotides will be considered in the description. It should be obvious to a person skilled in the art that nucleosides can also be modified in a corresponding manner and be converted into corresponding reactions. In principle, all conventional nucleotide variants suitable as substrate for nucleotide-accepting enzymes can serve as nuc-component of the nuc-macromolecule, so that both natural and modified nucleotides (nucleotide analogues) are suitable for the nuc-component. For modified nucleotides, base, sugar or phosphate moieties can be modified, 3 , Many examples of nucleotide modifications are known to those skilled in the art (Advanced Organic Chemistry of Nucleic Acids, 1994, Shabarova, ISBN 3-527-29021-4, "Nucleotide Analogs" Scheit, 1980, ISBN 0-471-04854-2, "Nucleosides and Nucleic Acid Chemistry", Kisakürek 2000, "Anti-HIV Nucleosides" Mitsuya, 1997, "Nucleoside analogs in cancer therapy ", Cheson, 1997) in the text, further examples of the modifications of the nucleotides are given.

The Nuc component preferably has a base component (base), a Sugar component (sugar) and a phosphate component (phosphate).

1.3.3.1.1 Variations on the phosphate

In an embodiment the nuc-component is a nucleoside. In another embodiment the nuc-component is a nucleoside monophosphate. In one another embodiment the nuc-component is a nucleoside diphosphate. In another embodiment the nuc-component is a nucleoside triphosphate. Also higher phosphate derivatives (Tetraphosphate, etc.) can be used.

The mentioned phosphate modifications can as natural Nucleoside triphosphates at the 5'-position or at other positions of the sugar part of the nucleotide sit, for example at 3 'position.

1.3.3.1.2 Variations at the base

The Nuk component can be a naturally occurring in the nucleic acids Nucleotide (with one of the bases adenine, guanine, thymine, Cytosine, uracil, inosine), or a modified base, e.g. 7-deazaadenine, 6-thio-adenine, included, literature s. above..

1.3.3.1.3 Variations on the sugar

Either Ribose as well as 2'-deoxyribose or 2 ', 3'-Dideoxyribose can backbone forming the nuc-components. Also on the 3'-hydroxyl modified ribose, or 2'-deoxyribose can be used. In the application, 2'-deoxynucleotides, for example, 2'-deoxyuridine triphosphate, 2'-deoxycytidine triphosphate, 2'-deoxyadenosine triphosphate, 2'deoxyguanosine triphosphate referred to as dUTP, dCTP, dATP and dGTP.

1.3.3.1.4 Coupling of NT and linker

The Nuk component is connected to the linker at a coupling site. The coupling site of the linker to the nuc-component may be at the base, on the sugar (ribose or deoxyribose), or on the phosphate part are located.

The Connection between the linker component and the nuk component is preferably covalent.

If the coupling site is at the base, it is preferably at positions 4 or 5 at pyrimidine bases and at positions 6,7,8 at the purine bases. At the sugar can the positions 2 ', 3 ', 4' or 5 'the coupling points represent. The coupling to the phosphate groups can be at the alpha, beta, or gamma phosphate group. Examples of the coupling site at the Base are in Short WO 9949082, Balasubramanian WO 03048387, Tcherkassov WO 02088382 (see also commercially available nucleotides (Amersham, Roche), at the ribose in Herrlein et al. Helvetica Chimica Acta, 1994, V. 77, p. 586, Jameson et al. Method in Enzymology, 1997, V. 278, p. 363, Canard US. Pat. 5798210, Kwiatkowski US Pat. No. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642., to phosphate groups in Jameson et al. Method in Enzymology, 1997, V. 278, p. 363.

The Position of the coupling point hangs from the field of application of NT macromolecules. For example for the Labeling of nucleic acids, at the nucleic acid strand should remain, preferably coupling sites on the sugar or on the base used. The coupling to the gamma or beta phosphate groups can be done, for example, when the mark during installation of the nuc macromolecule shall be.

The Connection between the nuc-component and the linker component over a coupling unit (L) which is a part of the linker component.

The Connection between the nuc-component and the linker can be in one embodiment resistant be, e.g. at temperatures up to 130 ° C, for pH ranges between 1 and 14, and / or resistant to hydrolytic enzymes (e.g., proteases, esterases) be. In another embodiment the invention is the connection between the nuc-component and the linker under mild conditions fissile.

These fissile connection allows the removal of linker and marker components. This can be, for example in the methods of sequencing by the synthesis of importance such as Pyrosequencing, BASS (Canard et al., U.S. Patent 5,798,210, Rasolonjatovo nucleosides & nucleotides 1999, V.18 p.1021, Metzker et al. NAR 1994, V.22, p.4259, Welch et al. Nucleosides & nucleotides 1999, V.18, p.19) or single molecule sequencing, Tcherkassov WO 02088382. Your choice is not limited, unless under the conditions the enzymatic reaction remains stable, no irreversible disruption of Enzymes (e.g., polymerase) and under mild conditions can be split off. Under "mild Conditions "are to understand such conditions, for example, nucleic acid primer complexes do not destroy, whereby e.g. the pH preferably between 3 and 11 and the temperature between 0 ° C and a temperature value (x). This temperature value (x) depends on the Tm of the nucleic acid primer complex (Tm is the melting point) and is used, for example, as Tm (nucleic acid-primer complex) minus 5 ° C calculated (e.g., Tm is 47 ° C, then the maximum temperature is 42 ° C; under these conditions Ester, thioester, acetals, phosphoester, Disulfide compounds and photolabile compounds as cleavable compounds).

Preferably belongs said cleavable compound to chemically or enzymatically cleavable or photolabile compounds. As examples of chemically cleavable Groups are ester, thioester, disulfide, acetal compounds preferred (Short WO 9949082, "Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993 CRC Press Inc., Herman et al. Method in Enzymology 1990 V.184 p.584, Lomant et al. J. Mol. 1976 V.104 243, "Chemistry of carboxylic acid and esters "S.Patai 1969 Interscience Publ.). Examples of photolabile compounds can can be found in the following references: Rothschild WO 9531429, "Protective groups in organic synthesis "1991 John Wiley & Sons, Inc., V. Pillai Synthesis 1980 p.1, V. Pillai Org. Photochem. 1987 V.9 p.225, dissertation "New photolabile protecting groups for light-directed oligonucleotide synthesis "H.Giegrich, 1996, Konstanz, Dissertation" Neue photolabile Protecting groups for light-directed oligonucleotide synthesis "S.M.Bühler, 1999, Konstanz) ..

1.3.3.1.5 Number of linked Nuc-components

In an embodiment According to the invention, only one nuc-component is coupled per one nuc-macromolecule. In another embodiment According to the invention, several nuc-components are coupled per nuc-macromolecule. Several nuc components can be uniform or different, for example, average 2 to 5, 5 to 10, 10 to 25, 25 to 50, 50 to 100, 100 to 250, 250 to 500, 500 to 1000 nuc-components per one nuk macromolecule coupled could be.

1.3.3.2 Linker component

The Designation Linker or linker component becomes synonymous in the application used and refers to the entire structural section of the nuc macromolecule between the nuk component and the marker component.

1.3.3.2.1 Linker components

• 1) Kopplungseinheit L
• 2) wasserlösliches Polymer
• 3) Kopplungseinheit T

The linker component is part of the nuclomor molecule that lies between the respective nuc-component and the marker component. The linker component preferably has the following components in its structure:

• 1) coupling unit L
• 2) water-soluble polymer
• 3) coupling unit T

The Division of the linker component into individual components is pure functional and intended only to illustrate the structure serve. Depending on the perspective, individual structures of the Linkers calculated on the one hand or on the other functional component become.

The coupling unit L has the function of connecting the linker component and the nuk component. Their structure is not limited as long as the nuc macromolecule as a substrate of the enzymes ak is seized. Preference is given to short, unbranched compounds, up to max. 20 atoms in length. The particular structure of the coupling unit L depends on the coupling site of the linker to the nucleotide and of the respective polymer of the linker. Some examples of the coupling units L are given in Examples 1 to 33. Many conventionally modified nucleotides carry a short linker, these short linkers serve as further examples of the coupling unit L, eg short linkers on the base: Short WO 9949082, Balasubramanian WO 03048387, Tcherkassov WO 02088382 (see also commercially available nucleotides (Amersham, Roche) Helvetica Chimica Acta, 1994, V. 77, p 586, Jameson et al., Method in Enzymology, 1997, V. 278, p 363, Canard US Pat. Kwiatkowski US Pat. No. 6,254,575, Kwiatkowski WO 01/25247, Parce WO 0050642., short linkers to phosphate groups in Jameson et al., Method in Enzymology, 1997, V. 278, p 363. Still further examples of the coupling unit L are given below :
R ₆ -NH-R ₇ , R ₆ -OR ₇ , R ₆ -SR ₇ , R ₆ -SS-R ₇ , R ₆ -CO-NH-R ₇ , R ₆ -NH-CO-R ₇ , R ₆ -CO-OR ₇ ,
R ₆ -O-CO-R ₇ , R ₆ -CO-SR ₇ , R ₆ -S-CO-R ₇ , R ₆ -P (O) ₂ -R ₇ , R ₆ -Si-R ₇ , R ₆ - (CH ₂ ) _n -R ₇ ,
R ₆ - (CH ₂ ) _n -R ₇ , R ₆ -A- (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -BR ₇ ,
R ₆ - (CH = CH-) _n -R ₇ , R ₆ - (A-CH = CH-) _n -R ₇ , R ₆ - (CH = CH-B-) _n -R ₇ ,
R ₆ -A-CH = CH- (CH ₂ -) _n -R ₇ , R ₆ - (- CH = CH-CH ₂ ) _n -BR ₇ , R ₆ - (- CH = CH-CH ₂ -CH ₂ ) _n -BR ₇ ,
R ₆ - (C≡C-) _n -R ₇ , R ₆ - (AC≡C-) _n -R ₇ , R ₆ - (C≡CB-) _n -R ₇ ,
R ₆ -AC≡C- (CH ₂ -) _n -R ₇ , R ₆ - (- C≡C-CH ₂ ) _n -BR ₇ , R ₆ - (- C≡C-CH ₂ -CH ₂ ) _n -BR ₇ ,
wherein R _{6 is} the nuc-component, R _{7 is the} polymer and A and B include the following structural elements: -NH-, -O-, -S-, -SS-, -CO-NH-, -NH- CO, -CO-O-, -O-CO-, -CO-S-, -S-CO-, -P (O) ₂ -, -Si-, - (CH ₂ ) _n -, a photolabile group where n - is 1 to 5

The Coupling unit L is on a side with the Nuk component, on the other covalently bonded to polymer. The type of coupling depends on the type of polymer. In a preferred form, the polymer has reactive groups, for example NH 2 (amino), OH (hydroxy), SH (mercapto), COOH (carboxy), CHO (aldehyde), acrylic or maleimide, Halogen groups. Such polymers are commercially available (e.g., Fluka). Some variants for the coupling of polymers to the coupling units are under Examples 1 to 33 indicated.

The water-soluble Polymer forms the major portion in a preferred embodiment the linker component. It is a polymer, preferably hydrophilic, consisting of the same or different monomers. Examples for suitable Polymers make polyethylene glycol (PEG), polysaccharides, polyamides (e.g. Polypeptides), polyphosphates, polyacetates, poly (alkylene glycols), Copolymers of ethylene glycol and propylene glycol, poly (olefinic Alcohols), poly (vinylpyrrolidones), poly (hydroxyalkylmethacrylamides), Poly (hydroxyalkyl methacrylates), poly (x-hydroxy acids), Polyacrylic acid and their derivatives, poly-acrylamides in their derivatives, poly (vinyl alcohol), Polylactate acid, polyglycolic acid, Poly (epsilon-caprolactone), poly (beta-hydroxybutyrate), poly (beta-hydroxyvalerate), Polydioxanone, poly (ethylene terephthalate), poly (malic acid), poly (tartronic Acid), Poly (ortho ester), polyanhydrides, polycyanoacrylates, poly (phosphoester), Polyphosphazenes, hyaluronidates, polysulfones.

This Polymer may or may not be branched. This polymer can be made consist of several sections of different lengths, each one Section consists of the same monomers and monomers in different Sections are different. A specialist should be obvious appear that for a macromolecular linkers usually only an average mass can be determined so that the information to the molecular weights ("macromolecules, chemical structure and Syntheses ", volume 1, 4, H. Elias, 1999, ISBN 3-527-29872-X). That's why for nuc macromolecules no exact Mass be made.

1.3.3.2.2 Linker length

The linker length is between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 to 100,000 atoms, thus the average Linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 to 100,000 angstroms (measured on a potentially maximally stretched molecule).

Some Sections of the linker can contain rigid areas and other sections can be flexible areas contain.

It will be apparent to those skilled in the obvious that the listed linker lengths a considerably larger molecule size and mass can have, as the respective nuc-component itself.

1.3.3.2.3 Linker coupling in a nuc macromolecule

Of the Linker is on one side to the nuk component and on the other Bound to the marker component. To do this, the linker has its end coupling units that perform this function. The connection with the nuk components was discussed above. The link between the linker and the marker components is via coupling unit T. Their structure is not limited as long as the nuc macromolecule as a substrate is accepted by the enzymes. Preferred are short, unbranched Connections, up to max. 20 atoms in length. The respective structure the coupling unit T hangs from the coupling site to the marker component and from the respective one Polymer of the linker. The coupling unit T is with the polymer covalently linked. The type of coupling depends on the type of polymer from. In preferred form, the polymer has reactive ends Groups, for example NH 2 (amino), OH (hydroxy), SH (mercapto), COOH (carboxy), CHO (aldehyde), acrylic or maleimide, halogen groups. Such polymers are commercially available (e.g., Fluka). Some examples for the coupling units L are given in Examples 1 to 33, Further examples of the chemical and affine couplings s. in the literature: "Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993," Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996.

The linker may also contain other functional groups or sections, for example one or more cleavable groups under mild conditions ( 9 ), one or dye molecules, s. Examples 22, 23, 24, 31.

A fissile connection within the linker allows removal of one Part of the linker and the marker component. After the split remains a linker residue at the nuc-component, examples of cleavable compounds are specified in section 1.3.3.1.4.

1.3.3.3 Marker component

The Marker component can have different structures. The structures In particular, they are not limited as long as they have the substrate properties the nuk components for enzymes not completely cancel. This structure preferably has a signaling or a signal-switching function. The marker may be different Have functions, such as structural, antitoxic or Affine function (for example, in medicines or Zuberetungen).

1.3.3.3.1 The composition the marker component

The marker can consist of a macromolecule ( 4a ) or a group of identical or different structural units (marker units) with signaling or signal-transmitting function. These units may be molecules of low molecular mass, eg less than 2000 Da, or even macromolecules themselves. In this case, the number of signaling or signal-transmitting units, which are combined to form a marker components, is between 1 and 2, 2 and 5, 5 and 20, 20 and 50, 50 and 100, 100 and 500, 500 and 1000. The units are bound to a macromolecular framework, the core components of the marker ( 4b , C). This core component connects the units to each other and connects to one or more nuc-linker components ( 5 ).

1.3.3.3.2 Structure of the signaling or the signal-transmitting units of the marker

The structural marker units can selected from the following groups become:

1.3.3.3.2.1 Structures with low molecular weight:

Biotin molecules, hapten molecules (eg digoxigenin), radioactive isotopes (eg P ³² , J ¹³¹ ), or their compounds, rare earths, dyes, fluorescent dyes (eg Molecular Probes, Inc.) with the same or different spectral properties, dyes, which are FRET among each other.

Also chemically reactive groups, such as amino, mercapto, aldehyde, iodoacetate, acrylic, dithio, thioester compounds can serve as signal-transmitting structural units ( 6a ). After incorporation, these reactive groups can be modified with signaling elements, such as, for example, dyes having corresponding reactive groups (for example NHS esters, mercapto, amino groups) ( 6b ). The basics for choosing a suitable pair are presented in "Chemistry of protein conjugation and crosslinking" Shan S. Wong 1993.

In a special embodiment He feels the combination of a macromolecular linker component and Already meets the requirements of a low molecular weight marker present invention. Such compounds are also subject matter of this invention, since they are used both as intermediates for the chemical Synthesis of nuc macromolecules with a macromolecular marker, e.g. dUTP-PEG-biotin, as well independent can be used in the enzymatic reactions, such as for example, with only one dye labeled nucleotides. When Dyes can different fluorescent dyes occur, their choice is not limited, as long as they do not significantly affect the enzymatic reaction. Examples for dyes Rhodamine (Rhodamine 110, tetramethylrhodamine), cyanine dyes (Cy2, Cy3, Cy5, Cy7), Coumarins, Bodipy, Fluorescein, Alexa Dyes. Many dyes are commercially available, for example from Molecular Probes Europe, Leiden, Netherlands (hereinafter referred to as Molecucular Probes) or Sigma-Aldrich-Fluka (Taufkirchen, Germany). examples for the synthesis of a nuc macromolecule with a low molecular weight Marker is given in Example 19, 20, 23, 36, 37, 38.

To the incorporation of the nucleotide portion in the nucleic acid is the linker coupled dye at a large distance from the nucleic acid strand, what to reduce the influences of the nucleobases on the fluorescence properties of the dye leads. Fluorescence yields individual Dyes are thus less of the local composition the nucleic acid sequence affected which leads to uniform signal intensity.

1.3.3.3.2.2 Structures high mass (macromolecules)

1.3.3.3.2.2.1 Nanocrystals, e.g. Quantum dots. This can be done in a marker component Quantum Dots used with the same or different spectral properties US Pat. No. 6,322,901, US Pat. No. 6,423,551, US Pat. No. 6,251,303, US Pat. US Pat. 5,990,479).

1.3.3.3.2.2.2 Nano or micro-particles, the largest dimensions of these particles of 1nm up to 2nm, from 2nm to 5nm, from 5nm to 10nm, from 10nm to 20nm, from 20nm to 50nm, from 50nm to 100nm, from 100nm to 200nm, from 200nm to 500nm, from 500nm to 1000nm, from 1000nm to 5000nm. The material of the particles can For example, pure metals such as gold, silver, aluminum (for example Particles with surface plasmon resonance), - protein Au conjugates: J. Anal.Chem. 1998, v. 70, p. 5177, - nucleic acid-Au conjugates: J. Am. Chem. Soc. 2001, V. 123, p.5164, J. Am. Chem. Soc. 2000, V. 122, p. 9071, Biochem. Biophys. Res. Commun 2000, V. 274, p. 817, Anal. Chem. 2001, V. 73, p. 4450), - latex (e.g., latex nanoparticles, Anal. Chem., 2000, v. 72, p. 1979) - plastic (Polystyrene), - paramagnetic Compounds / Mixtures Zhi ZL et al. Anal Biochem. 2003 Jul 15; 318 (2): 236-43, Dressman D et al. Proc Natl Acad Sci U S A. 2003 Jul 22; 100 (15): 8817-22 ,, - Metal particles, - Magnetic Compounds / Mixtures Jain KK. Expert Rev Mol Diagn. 2003 Mar; 3 (2): 153-61, Patolsky F et al. Angew Chem Int Ed Engl. 2003 May 30; 42 (21): 2372-2376, Zhao X et al. Anal Chem. 2003 Jul 15; 75 (14): 3144-51, Xu H et al. J Biomed Mater Res. 2003 Sep 15; 66A (4): 870-9, JOSEPHONE Lee et al. US2003092029, KLICHE KAY-OLIVER et al. WO0119405.

1.3.3.3.2.2.3 Protein molecules,

• from the following groups: enzymes (e.g., peroxidase, alkaline Phosphotase, urease, beta-galactosidase), - fluorescent proteins (e.g., GFP), antigen-binding proteins (e.g., antibodies, tetramers, Affibodies (Nord et al., Al Nature Biotechnology, V. 15 (S.772-777)). or their components (e.g., Fab fragments), nucleic acid binding agents Proteins (e.g., transcription factor)

1.3.3.3.2.2.4 Nucleic acid chains,

• the nucleic acid chains including the group, oligonucleotides, modified oligonucleotides, wherein the nucleic acid chains have a length of from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 200, from 200 to 500, from 500 to 1000, 1000 to 5000, from 5,000 to 10,000, from 10,000 to 100,000 nucleotides. DNA, RNA, PNA molecules can be used. Nucleic acid chains can carry additional modifications, such as, for example, free amino groups, dyes and other signaling molecules, eg macromolecular substances, such as enzymes or nanocrystals ( 7a , C) (MWG-Biotech).

Further examples of macromolecules or macromolecule complexes that can be used in the present invention as units in the signal amplification marker component are disclosed in U.S. Pat US 4882269 . US 4687732 , WO 8903849, US 6017707 described.

1.3.3.3.3 The core component the marker component

The core component has the function of binding several structural elements of the nuc macromolecules. Preferably, several nucl linker components or multiple label moieties are coupled to the core component, s. 5 ,

1.3.3.3.3.1 Components

In an embodiment The core component consists of a water-soluble polymer, it can the polymer consist of the same or different monomers.

Examples of the core component include derivatives of the following polymers: polyamides (eg, polypeptides), polyacrylic acid, polyacrylamides, polyvinyl alcohols, nucleic acids, proteins. These polymers may be linear, globular, eg, streptavidin or avidin, or branched, eg dendrimers ( 8a ). Crosslinked soluble polymers, such as, for example, crosslinked polyacrylamides (crosslinker bisacrylamide in combination with polyacrylamide), are used.

The Kern-Komponennte has several coupling points to the other elements can be bound e.g. structural marker units or nuc-linker component.

For example Poly-lysine molecules have multiple free amino groups to which multiple dye molecules, biotin molecules, hapten molecules or nucleic acid chains can be coupled. Poly-lysines have different molecular weights, e.g. 1000-2000, 2000-10000, 10000-50000 There you can for sale be acquired.

As a further example of the core component serve nucleic acid strands, wherein the nucleic acid chains have a length of 10 to 20, from 20 to 50, from 50 to 100, from 100 to 200, from 200 to 500, from 500 to 1000, 1000 to 5000, of 5000 to have 10,000 nucleotides. These nucleic acids serve as binding partners for sequence-complementary marker units ( 7b ).

In a further embodiment if the core component consists of a dendrimer, e.g. polypropylenimines, Polyaminoamines. examples for Other dendrimers are known: Cientifica "Dendrimers", 2003, Technology white papers No. 6, Klajnert et al. Acta Biochimica Polonica, 2001, v. 48, p. 199, Manduchi et al. Physiol.

Genomics 2002, V.10, p.169, Sharma et al. Electrophoresis. 2003, v. 24, p. 2733, Morgan et al. Curr Opin Drug Discov Devel. 2002 Nov; 5 (6): 966-73, Benters et al. Nucleic Acids Res. 2002 Jan 15; 30 (2): E10, Nilsen et al. J Theor Biol. 1997 Jul 21; 187 (2): 273-84. Many dendrimers are commercially available (Genisphere, www.genisphere.com, Chimera Biotech GmbH).

Further Combinations for the core components of the components shown above are obvious to a person skilled in the art.

1.3.3.3.3.2 Coupling of the Marker units to the core component

structural Marker units can to the core component or to the linker component through a covalent bond, for example about a cross-linker (Chemistry of protein conjugation and cross-linking, S. Wang, 1993, ISBN 0-8493-5886-8, "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2), or over an affine coupling, for example, biotin-streptavidin compound or hybridization of nucleic acid strands or Antigen-antibody interaction ( "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2).

The Coupling of marker units to the core component takes place in an embodiment already during the synthesis of nuc macromolecules.

In another embodiment, first the chemical synthesis of nuc-macromolecules takes place, in which the marker component consists only of the core component. The coupling of marker units to the core component takes place only after the incorporation of nuc-macromolecules into the nucleic acid chain. Due to a large number of potential binding sites on the core part, the likelihood of binding is Marker units to the core component and thus to the incorporated nuc-component much larger compared to conventional nucleotide structures. The coupling chemistry in each case depends on the structure of the marker units and the structure of the core component.

covalent Coupling: The link between the marker units and the core component can be found in an embodiment resistant (Example 33), e.g. at temperatures up to 100 ° C, for pH ranges between 3 and 12, and / or resistant to hydrolytic enzymes (e.g., esterases). In another embodiment of the invention the connection between the nuc-component and the linker under mild Conditions fissile.

Examples for the Coupling of nucleic acids on dendrimers (corresponds to a coupling of marker units to a core component) are e.g. in Shchepinov et al. Nucleic Acids Res. 1999 Aug 1; 27 (15): 3035-41, Goh et al. Chem Commun (Camb). 2002 Dec 21; (24): 2954.

1.3.3.3.3.3 Coupling between Linker and marker

In one embodiment, the nuk linker component is bound directly to the signaling or signal-mediating marker unit of the marker component, 4a ,

In another embodiment, one or more nucl linker moieties are attached to the core component of the label, 5d ,

The Connection between the linker and the core component or the Linker and the marker component depends on the particular structure the marker units or the structure of the core component. The Binding can be covalent as well as affine. "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2.

covalent Coupling: The connection between the marker units and the core component can in one embodiment resistant be, e.g. at temperatures up to 130 ° C, for pH ranges between 1 and 14, and / or resistant to hydrolytic enzymes (e.g., proteases, Esterases). In another embodiment of the invention the connection between the nuc-component and the linker below mild conditions fissile.

1.3.3.3.4 The ratio of Nuc components in a nuc macromolecule

In a nuc macromolecule can 1-1000 Nuk components, s.o. The distribution can be Nuk components in a nuc-macromolecule population are, and do not have to vary necessarily represent a constant value for each nuc-macromolecule.

In an embodiment all have nuc macromolecules an equal number of nuc components per a nuc macromolecule. For example, you can maximum for one strepavidin molecule 4 biotin molecules be bound at saturating Concentration of nucl linker components, a uniform population on nuc macromolecules arises.

In another embodiment have the nuc macromolecules population has a defined average number of nuclei Components per nuc-macromolecule, in the Population itself, however, finds a distribution of actual Occupation of the nuc macromolecules with Nuk components instead. The distribution information of nuc components per nuc-macromolecule in this case represent an average. Such nuc-macromolecules may be, for example be used when the nuc-component has a terminating Has property.

1.3.3.3.5 The ratio of Marker units in a nuc macromolecule

In a nuc macromolecule can 1-1000 marker units occur, s.o. The distribution can be vary from marker units in a nuc-macromolecule population and does not have to necessarily represent a constant value for each nuc-macromolecule.

In an embodiment all have nuc macromolecules an equal number of marker units per one nuc macromolecule. For example can per one strepavidin molecule a maximum of 4 biotin molecules be bound, avidin biotin technology, Methods in Enzymology v.184, 1990.

In another embodiment have the nuc macromolecules a population a defined average number of marker units per nuc-macromolecule, in the population itself, however, finds a distribution of the actual Occupation of the nuc macromolecules held with marker units. At saturating concentrations The synthesis of marker components is becoming increasingly uniform Occupation of the nuc macromolecules held with marker units.

Such Nuc-macromolecules are for example Methods of importance in which fluctuations in the signal intensity within one Nuc-macromolecule population are negligible.

So plays for example in areas, where it concerns the qualitative proof is the exact number of marker units per nuc-macromolecule a subordinate Role. In such cases the presence of a stable signal is important. examples for such methods are single molecule sequencing methods.

It It should be apparent to one skilled in the art that the recited marker components are a significant larger molecule size and mass have, as the respective nuk component itself. Furthermore, should other examples of macromolecular marker components to a person skilled in the obvious.

1.3.3.3.6 Substrate properties the nuc macromolecules

The Nuk component can serve as a substrate for different enzymes. For example, the nuc-component is used in this embodiment as nucleoside triphosphate, as a substrate for a polymerase so that the Nuk component in a growing strand by a polymerase can be incorporated and thus the entire nuc-macromolecule to the Strand is covalently coupled.

Further examples for Enzymes are kinases, phosphorylases, transferases.

The Determine substrate properties of the nuc-component (s) the substrate properties of the nuc macromolecules. So can the nuk component serve as a terminator, so that only a single nuc-macromolecule incorporated can be. In another embodiment, the nuc-component is used as a reversible terminator, which controlled the implementation of a gradual Extension reaction allowed, such as. in Tcherkassov WO 02088382 is shown.

1.3.3.3.7 Function of the marker

The Macromolecular marker component may be in one embodiment a signaling, signal-transmitting, catalytic or affine Have function.

at contains the signaling function the marker component constituents already during the chemical synthesis to nuc-macromolecules are bound, s. example 33rd

at the signal-transmitting function carries the marker component components, the first through a reaction with signaling molecules their Develop signal properties, s. Example 32.

For example can several biotin molecules, e.g. 100; form the marker part. After incorporation of the nuc-macromolecules takes place a detection reaction with modified streptavidin molecules. In In another example, the nucleic acid chains have the signal-transmitting Function. After incorporation of nuc-macromolecules, hybridization occurs of uniform oligonucleotides with detectable units, e.g. Fluorescent dyes (MWG Biotech) to the marker component. In one further examples have amino or mercapto groups, for example 50 amino groups per marker, the signal-transmitting function. After this Incorporation of nuc macromolecules into the nucleic acid chain a chemical modification with reactive components, e.g. with dyes, such as for incorporated allyl-amino-dUTP described, Diehl et al. Nucleic Acid Research, 2002, V. 30, No. 16 e79.

In another embodiment the macromolecular marker component has a catalytic function (in the form of an enzyme or ribozyme). It can be different enzymes can be used, e.g. Peroxidase or alkaline phosphatase. thanks the coupling to the nuc-component, the respective enzyme by the incorporation of nuc macromolecules to the nucleic acid strand covalently bound.

In a further embodiment the macromolecular marker component has an affinity functionality to one another molecule. examples for such markers form streptavidin molecules or nucleic acid chains, Example 30 or 32.

1.3.4. Low Molecular Marker - for State of the art Labeling of nucleotides, for example, with one or two biotin molecules or two dye molecules, one or two hapten molecules (e.g., digoxigenin).

1.3.5. Conventionally modified nucleotide - a nucleotide with a linker (average length between 5 and 30 atoms) and a marker. Usually wearing a conventional modified nucleotide comprises a low molecular weight marker, e.g. one Dye or biotin molecule.

To Demonstration of the fact that a simple combination of a conventionally modified nucleotide with a macromolecular marker to abolish the substrate properties the nucleotide leads nucleotides represented in this application, although a macromolecular Marker but a short linker with one of an average length of Carry 5 to 30 atoms. Even such nucleotides are considered conventional denotes modified nucleotides. Alone the combination between a conventionally modified nucleotide and a macromolecular Marker is not enough according to the invention to meet requirements of the definition of nuc macromolecules.

in the By contrast, nuc macromolecules are clearly unique through a combination from one or more nuc-components, corresponding to one or more defined by several long linkers and a marker.

1.3.6. polymerase

In choosing the polymerase, the type of immobilized nucleic acid used (RNA or DNA) plays a crucial role:
If RNA is used as substrate (eg mRNA) in the sequencing reaction, commercial RNA-dependent DNA polymerases can be used, eg AMV reverse transcriptase (Sigma), M-MLV reverse transcriptase (Sigma), HIV reverse transcriptase without RNAse- Activity. For certain applications, reverse transcriptases may be largely free of RNAse activity ("Molecular cloning" 1989, Ed. Maniatis, Cold Spring Harbor Laboratory), eg in mRNA migation for hybridization experiments.

If DNA as a substrate (e.g., cDNA) are useful as polymerases in principle all DNA-dependent DNA polymerases with or without 3'-5 'exonuclease activity (DNA replication "1992 ed. A.Kornberg, Freeman and company NY), e.g. modified T7 polymerase type "Sequenase Version 2 "(Amersham Pharmacia Biotech), Klenow fragment of DNA polymerase I with or without 3'-5 'exonuclease activity (Amersham Pharmacia Biotech), polymerase beta of various origins (Animal Cell DNA Polymerases "1983, Fry M., CRC Press Inc., commercially available from Chimerx) thermostable Polymerases such as Taq polymerase (GibcoBRL), proHA DNA polymerase (Eurogentec), Vent, Vent exo minus, Pfu etc.

Also DNA-dependent RNA polymerases can can be used, e.g. E. coli RNA polymerase, T7 RNA polymerase, SP6 RNA Polymerase.

In The registration will be DNA-dependent DNA polymerases as examples of considered all polymerases.

1.3.7. Fissile connection - One under mild conditions, fissile compound. This connection can represent a section in the linker and can be at or be cleavable in several places. It can be a chemical cleavable compound such as a disulfide, an ester, a Acetal, a thioester compound (Short WO 9949082, Tcherkassov WO 02088382). It can also be a photochemically fissile Compound as shown in (Rothschild WO 9531429). It may also be an enzymatically cleavable compound (for example a peptide or polypeptide linkage, Odedra WO 0192284), cleavable by Peptidases, a poly- or oligosaccharide bond, cleavable by Disaccharidases), wherein the cleavage by a specific Enzyme may take place between certain monomers of the cleavable sites.

Several examples of scissile compounds are known. The coupling of such a compound is described, for example, in (Tcherkassov 02088382, Metzker et al., Nucleic Acid Research 1994, V.22, p. 4259, Canard et al. Gene, 1994, V. 148, 1, Kwiatkowski US Pat. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642.). A cleavable compound may be part of the linker or may form the coupling site of the linker to the nucleotide, or the linkage between the linker moiety and the macroser moiety, or the linkage between the label moieties and the core moiety.

1.3.8 DNA - deoxyribonucleic acid different Of origin and different length (e.g., oligonucleotides, Polynucleotides, plasmids, genomic DNA, cDNA, ssDNA, dsDNA)

1.3.9 RNA - ribonucleic acid

01/03/10 dNTP - 2'-deoxynucleoside triphosphates, Substrates for DNA polymerases and reverse transcriptases, e.g. dATP, dGTP, dUTP, dTTP, dCTP.

01/03/11 NTP - ribonucleoside triphosphates, Substrates for RNA polymerases, UTP, CTP, ATP, GTP.

01/03/12 Abbreviation "NT" is also used in the length specification a nucleic acid sequence used, e.g. 1,000 NT. In this case, "NT" stands for nucleoside monophosphates.

in the Text becomes abbreviations the majority formed by using the suffix "s", "NT" is for example for "nucleotide", "NTs" stands for several Nucleotides.

1.3.13 NSK - Nucleic Acid Chain. DNA or RNA

01/03/14 Total sequence - total of all sequences in the approach, it may originally be one or more NSKs exist. The total sequence may be parts or equivalents another sequence or sequence populations (e.g. mRNA, cDNA, plasmid DNA with insert, BAC, YAC) and from one or more come from different species.

01/03/15 NSKF - nucleic acid chain fragment (DNA or RNA) corresponding to part of the total sequence, NSKFs - nucleic acid chain fragments. The sum of the NSKFs is one equivalent to the overall sequence. The NSKFs can For example, be fragments of DNA or RNA total sequence, the arise after a fragmentation step.

01/03/16 Primer binding site (PBS) - part the sequence in the NSK or NSKF to which the primer binds.

01/03/17 Reference sequence - one already known sequence to which the deviations in the examined Sequence or in the sequences to be examined (for example total sequence) be determined. As reference sequences can be accessed in databases Sequences are used, e.g. from the NCBI database.

1.3.18 Tm - melting temperature

01/03/19 Steric Obstacle: Sterically demanding group that is through their chemical structure has the properties of this group coupled nucleotides changed so that this is not due to a polymerase in an extension reaction can be installed one after the other.

1.3.20 PNA peptides Nucleic acid

2. Detailed description:

The invention describes a new class of modified nucleotides. 1. Aspect of the invention relates to macromolecular compounds having the structure: (Nuk linker) _n marker in which:
Nuk - a nucleotide or a nucleoside is (Nuk component)
Linker - a linker component that includes the following components:
a) coupling unit L - a part of the linker, which represents the connection between Nuk and the linker radical
b) Polymer - a part of the linker containing a water-soluble polymer with an average length between 100 and 10,000 atoms is
c) Coupling unit T - a part of the linker that represents the link between the linker residue and the marker
Marker - a marker component is
n - is an integer between 1 and 100

Another aspect 2 of the invention relates to macromolecular compounds according to aspect 1, wherein the nuc-component includes the following structures ( 3A ):
In which:
Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications, where L is the link between the nuc-component and the linker Component represents (coupling unit L) and X is the coupling point of the coupling unit L at the base
R ₁ - is H
R ₂ - is independently selected from the group H, OH, halogen, NH ₂ , SH or protected OH group
R ₃ - is independently selected from the group H, OH, Hal, PO ₃ , SH, NH ₂ , OR _3-1 , P (O) _m -R _3-1 , NH-R _3-1 , SR _3-1 , Si-R _3-1 wherein R _{3-1 is} a chemically, photochemically or enzymatically cleavable group and m is 1 or 2.
R ₄ - is H or OH
R ₅ - is independently selected from the group OH, or a protected OH group, or monophosphate group, or diphosphate group or triphosphate group or polyphosphates

Another aspect 3 of the invention relates to macromolecular compounds according to aspect 1, wherein the nuc-component includes the following structures ( 3B ):
In which:
Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications capable of enzymatic reactions
R ₁ - is H
R ₂ - is independently selected from the group H, OH, Hal, NH ₂ , SH or protected OH group
R ₃ - is independently selected from the group OR _3-2 , P (O) _m -R _3-2 , where m is 1 or 2, NH-R _3-2 , SR _3-2 , Si-R _3-2 or, where R _{3-2 is} the coupling site of the
Coupling unit L to the nucleotide
Coupling unit L - the connection between the nuc-component and the linker component
R ₄ - is H or OH
R ₅ - is independently selected from the group OH, or a protected OH group, or monophosphate group, or diphosphate group or triphosphate group

Another aspect 4 of the invention relates to macromolecular compounds according to aspect 1, wherein the nuc-component includes the following structures ( 3B ):
In which:
Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications capable of enzymatic reactions
R ₁ - is H
R ₂ - is independently selected from the group H, OH, Hal, NH ₂ , SH or protected OH group
R ₃ - is independently selected from the group H, OH, Hal, PO ₃ , SH, NH ₂ , OR _3-1 ,
P (O) m R _3-1 , NH-R _3-1 , SR _3-1 , Si-R _3-1 wherein R _{3-1 is} a chemically, photochemically or enzymatically cleavable group and m is 1 or 2.
R ₄ - is H or OH
R ₅ - is independently selected from the group OR _5-1 -L, or P- (O) ₃ -R _5-1 -L (modified monophosphate group), or P- (O) ₃ -P- (O) ₃ -R _5-1 -L (modified diphosphate group)
or P- (O) ₃ -P- (O) _3- P- (O) ₃ -R _5-1 -L (modified triphosphate group), wherein R _{5-1 is} the coupling site of the coupling unit L to the nucleotide, and Coupling unit L is the connection between the nuc-component and the linker component.

Another aspect 5 of the invention relates to macromolecular compounds according to aspects 1 to 4, wherein coupling unit L includes the following structural elements:
R ₆ -NH-R ₇ , R ₆ -OR ₇ , R ₆ -SR ₇ , R ₆ -SS-R ₇ , R ₆ -CO-NH-R ₇ , R ₆ -NH-CO-R ₇ , R ₆ -CO-OR ₇ ,
R ₆ -O-CO-R ₇ , R ₆ -CO-SR ₇ , R ₆ -S-CO-R ₇ , R ₆ -P (O) ₂ -R ₇ , R ₆ -Si-R ₇ , R ₆ - (CH ₂ ) _n -R ₇ ,
R ₆ - (CH ₂ ) _n -R ₇ , R ₆ -A- (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -BR ₇ ,
R ₆ - (CH = CH-) _n -R ₇ , R ₆ - (A-CH = CH-) _n -R ₇ , R ₆ - (CH = CH-B-) _n -R ₇ , R ₆ - ( CH = CH-CH ₂ -B-) _n -R ₇ , R ₆ -A-CH = CH- (CH ₂ -) _n -R ₇ , R ₆ - (- CH = CH-CH ₂ ) _n -BR ₇ .
R ₆ - (C≡C-) _n -R ₇ , R ₆ - (AC≡C-) _n -R ₇ , R ₆ - (AC≡C-CH ₂ ) _n -R ₇ , R ₆ - (C≡ CB-) _n -R ₇ ,
R ₆ - (C≡C-CH ₂ -B-) _n -R ₇ , R ₆ -AC≡C- (CH ₂ -) _n -R ₇ , R ₆ - (- C≡C-CH ₂ ) _n - BR ₇ ,
R ₆ - (- C≡C-CH ₂ -CH ₂ ) _n -BR ₇
wherein R ₆ - is the nuc-component, R ₇ - is the linker radical and A and B independently include the following structural elements: -NH-, -O-, -S-, -SS-, -CO-NH-, -NH -CO-, -CO-O-, -O-CO-, -CO-S-, -S-CO-, -P (O) ₂ -, -Si-, - (CH ₂ ) _n -, a photolabile Group, where n - is 1 to 5

One Further aspect 6 of the invention relates to macromolecular compounds according to aspects 1 to 5, wherein the linker component is a water-soluble Polymer includes.

Another aspect 7 of the invention relates to macromolecular compounds according to aspect 6, wherein the linker component includes water-soluble polymers independently selected from the following group:
Polyethylene glycol (PEG), polysaccharides, dextran, polyamides, polypeptides, polyphosphates, polyacetates, poly (alkylene glycols), copolymers of ethylene glycol and propylene glycol, poly (olefinic alcohols), poly (vinyl pyrrolidones), poly (hydroxyalkyl methacrylamides), poly (hydroxyalkyl methacrylates) , Poly (x-hydroxy acids), polyacrylic acid, polyacrylamide, poly (vinyl alcohol).

One Further aspect 8 of the invention relates to macromolecular compounds according to aspects 1 to 7, wherein the linker component is an average Length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 to 100,000 atoms.

One Further aspect 9 of the invention relates to macromolecular compounds according to aspects 1 to 8, wherein a marker component is a signaling, has signal-transmitting, catalytic or affine function

One Another aspect of the invention relates to macromolecular compounds according to aspects 1 to 9, wherein a marker component of a structural Marker unit exists

One Further aspect 11 of the invention relates to macromolecular compounds according to aspects 1 to 9, wherein a marker component of several structural marker units bound to a core component consists

Another aspect 12 of the invention relates to macromolecular compounds according to aspects 10 or 11, wherein a structural marker unit independently includes one of the following structural elements:
Biotin, hapten, radioactive isotope, rare earth element, dye, fluorescent dye.

Another aspect 13 of the invention relates to macromolecular compounds according to aspects 10 or 11, wherein a structural marker unit independently includes one of the following elements:
Nanocrystals or their modifications, proteins or their modifications, nucleic acid chains or their modifications, particles or their modifications.

Another aspect of the invention relates to macromolecular compounds according to aspect 13, wherein a structural marker unit includes one of the following proteins:
Enzymes or their conjugates or modifications,
Antibodies or their conjugates or modifications,
Streptavidin or its conjugates or modifications,
Avidin or its conjugates or modifications

One Further aspect 15 of the invention relates to macromolecular compounds according to aspect 13, wherein a structural marker unit is one of following types of nucleic acid chains includes: DNA, RNA, PNA, where the length of nucleic acid chains between 10 and 10,000 nucleotides or their equivalents.

Another aspect of the invention pertains to macromolecular compounds of aspects 11-15, wherein the core component of the marker component includes independently one of the following elements: water soluble polymer independently selected from the group of: polyamides (eg polypeptides), polyacrylic acid and their derivatives, polyacrylamides and their derivatives, polyvinyl alcohols and their derivatives, nucleic acid chains and their derivatives, streptavidin or avidin and their derivatives, dendrimers, wherein individual polymers may be linear or branched or interlinked Another aspect 17 of the invention Macromolecular compounds according to aspects 1 to 9, 11 to 16, wherein the compound between a plurality of structural marker units and the core component is covalent or affine.

One Further aspect 18 of the invention relates to macromolecular compounds according to aspects 1 to 10, wherein the connection between a structural Marker unit and the linker is covalent or affine.

One Further aspect 19 of the invention relates to macromolecular compounds according to aspects 1 to 9, 11 to 17, wherein the connection between the core component and the linker is covalent or affine

One Further aspect 20 of the invention relates to macromolecular compounds according to aspects 1 to 19, wherein only one nuc-component with a coupled linker component bound to the marker component is, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms.

One Further aspect 21 of the invention relates to macromolecular compounds according to aspects 1 to 20, wherein only one nuc-component with a coupled linker component bound to the marker component is, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and the linker-fighting moment one or more compounds which can be cleaved under mild conditions includes.

One Further aspect 22 of the invention relates to macromolecular compounds according to aspects 1 to 21, wherein only one nuc-component with a coupled linker component bound to the marker component is, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and one or more parts of the nuc macromolecule so modified are that only a nuk component can be incorporated into a growing strand can.

One Further aspect 23 of the invention relates to macromolecular compounds according to aspects 1 to 19, wherein several Nuk components each have a Linker coupled to a marker components, the length of the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms.

One Further aspect 24 of the invention relates to macromolecular compounds Aspects 1 to 19, 23, wherein several Nuk components each have a Linker coupled to a marker components, with the length of the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and the respective linker one or more cleavable under mild conditions Includes connections.

One Further aspect 25 of the invention relates to macromolecular compounds Aspects 1 to 19, 23, 24, wherein several Nuk components each have a Linker coupled to a marker components, with the length of the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and a or several parts of the nuc macromolecule are so modified that only one Nuk component can be incorporated into a growing nucleic acid chain.

One Further aspect 26 of the invention relates to oligonucleotides or polynucleotides the at least one nuc-macromolecule according to aspects 1 to 25 per a nucleic acid chain lock in.

One Further aspect 27 of the invention relates to oligonucleotides or polynucleotides according to aspect 26, wherein oligo- or polynucleotides are RNA, DNA or PNA are whose length between 5 and 50,000 nucleotides.

One Further aspect 28 of the invention relates to a method for modification of nucleic acid chains, wherein for the Coupling nuc-macromolecules according to aspects 1 to 25 are used

• – mindestens eine Art der Nuk-Makromoleküle oder deren Zwischenstufen nach Aspekten 1 bis 25, wobei jede Art der Nuk-Makromoleküle eine für sie charakteristische Markierung besitzt
• – mindestens eine Population der Nukleinsäureketten,
• – mindestens eine Enzymart zur Kopplung von Nuk-Makromoleküle an die Nukleinsäureketten,

Another aspect 29 of the invention relates to a method according to aspect 28, wherein the modification is by an enzymatic coupling and the reaction mixture includes the following components:

• At least one kind of the nuc-macromolecules or their intermediates according to aspects 1 to 25, wherein each kind of the nuc-macromolecules has a characteristic marking for them
• At least one population of the nucleic acid chains,
• At least one type of enzyme for coupling nuc macromolecules to the nucleic acid chains,

• – mindestans eine Art der Nuk-Makromoleküle oder deren Zwischenstufen nach Aspekten 1-25, wobei jede Art der Nuk-Makromoleküle eine für sie charakteristische Markierung besitzt
• – mindestens eine Population der Nukleinsäureketten,
• – mindestens eine Enzymart zur Kopplung von Nuk-Makromoelküle an die Nukleeinsäureketten
• – mindestens eine weitere Art von Nukleosidtriphosphaten

Another aspect of the invention relates to a method according to aspect 28, wherein the modification is by an enzymatic coupling and the reaction mixture includes the following components:

• At least one type of nuc-macromolecule or its intermediates according to aspects 1-25, wherein each type of nuc-macromolecule possesses a characteristic marking for them
• At least one population of the nucleic acid chains,
• - At least one type of enzyme for the coupling of nuc-macromolecules to the nucleic acid chains
• - At least one other type of nucleoside triphosphates

One Further aspect 31 of the invention relates to a method according to aspects 29, 30, the type of enzyme being independent includes one of the following groups: DNA polymerase, RNA polymerase, Terminal transferase,

One Further aspect 32 of the invention relates to a method according to aspect 30, the "other Type of nucleoside triphosphates independently selected is from the group of: ribo-nucleoside triphosphates (ATP, GTP, UTP, CTP), from 2'-deoxyribonucleoside triphosphates (dATP, dUTP, dTTP, dCTP, dGTP) of 2 ', 3'-dideoxynucleoside triphosphates (ddATP, ddGTP, ddUTP, ddCTP, ddTTP).

One Further aspect 33 of the invention relates to a method according to aspect 32, the "other Type of nucleoside triphosphates are conventionally modified nucleotides with a label, this label being independent selected is from the group of: a fluorescent dye, a biotin, a Hapten, a radioactive element.

One Another aspect 34 of the invention relates to a method according to aspects 28 to 33, with at least two different populations nucleic acid chains present are

One Further aspect 35 of the invention relates to a method according to aspect 34, wherein at least one of the populations of nucleic acid chains a primer function and at least one of the nucleic acid chains has a template function.

One Further aspect 36 of the invention relates to a method according to aspect 28, wherein the modification is effected by a chemical coupling, wherein the coupling of the nuc-macromolecules to nucleic acid chains by Phosphoroamidite coupling takes place.

One Further aspect 37 of the invention relates to a method according to aspects 28 to 36, wherein Nuk macromolecules used in the labeling process Be the coupling of only a single Nuk component in the growing nucleic acid strand allow multiple installation by modifications to the Nuk component, and / or the linker component and / or the marker component prevented becomes.

One Further aspect 38 of the invention relates to a method according to aspect 37, wherein the further coupling is reversibly prevented.

One Further aspect 39 of the invention relates to a method according to aspect 37, wherein the further coupling is irreversibly prevented.

One Another aspect 40 of the invention relates to a method according to aspects 28 to 36, wherein Nuk macromolecules used in the labeling process Becoming a coupling of several Nuk component in the growing nucleic acid strand allow

One Another aspect 41 of the invention relates to a method according to aspects 28-40, wherein the participating in the reaction nucleic acid chains are coupled to a fixed phase and have addressable positions.

One Further aspect 42 of the invention relates to a method according to aspect 41, wherein the nucleic acid chains represent a uniform population

One Further aspect 43 of the invention relates to a method according to aspect 41, wherein the nucleic acid chains represent two or more different populations and each populations an addressable position on the solid phase Has

Another aspect 44 of the invention relates to a method according to aspects 41, 42, wherein the coupling Nuc-macromolecules are carried out on a population of uniform, solid-phase-fixed nucleic acid molecules, wherein the marker component of the Nuk macromolecule remains after coupling on the extended nucleic acid strand and is not separated off.

One Another aspect 45 of the invention relates to a method according to aspects 41.42, where the coupling of nuc-macromolecules to a population is uniform, takes place on the solid phase of fixed nucleic acid molecules, wherein the marker component or its individual components with or without linker component of the nuc macromolecule during the Coupling or after the coupling of the growing in the nucleic acid strand built-in Nuk component is separated.

One Another aspect 46 of the invention relates to a method according to aspects 41.43, wherein the coupling of nuc macromolecules in a reaction mixture parallel to two or more different populations the solid phase of fixed nucleic acid molecules takes place, these populations each different addressable positions on the fixed Phase and the marker component of the nuc macromolecule the coupling on the extended nucleic acid strand remains and is not separated.

One Another aspect 47 of the invention relates to a method according to aspects 41.43, wherein the coupling of nuc macromolecules in a reaction mixture parallel to two or more different populations the solid phase of fixed nucleic acid molecules takes place, these populations have different addressable positions at the solid phase and the marker component or its individual components with or without linker component of the nuc macromolecule during the Coupling or after the coupling of the nuc-component is separated.

One Further aspect 48 of the invention relates to a method according to aspects 41 to 47, wherein the addressable positions with nucleic acid molecules on the solid phase distributed as spots on a flat surface are, where per one spot nucleic acid molecules are uniform.

One Further aspect 49 of the invention relates to a method according to aspects 41 to 47, wherein the addressable positions with nucleic acid molecules to the globule or particles are attached, wherein per a bead nucleic acid molecules uniformly are.

One Another aspect 50 of the invention relates to a method according to aspects 41 to 47, wherein the addressable positions with nucleic acid molecules in a Multi vascular system, like microtiter plate or nanotiter plate or picotiter plate, are distributed, wherein in a vessel of the multi-vascular system the nucleic acid molecules uniform are.

• a) Bereitstellung mindestens einer Population an einzelsträngigen Nukleinsäureketten
• b) Hybridisierung von sequenzspezifischen Primern an diese Nukleinsäureketten, wobei extensionsfähige NSK-Primer-Komplexe entstehen
• c) Inkubation von mindestens einer Art der Nuk-Makromoleküle nach Aspekten 1 bis 25 zusammen mit einer Art der Polymerase nach Aspekt 31 mit den in Schritten a und b bereitgestellten NSK-Primer-Komplexen unter Bedingungen, die den Einbau von komplementären Nuk-Makromolekülen zulassen, wobei jede Art der Nuk-Makromoleküle eine für sie charakteristische Markierung besitzt.
• d) Entfernung der nicht eingebauten Nuk-Makromoleküle von den NSK-Primer-Komplexen
• e) Detektion der Signale von in die NSK-Primer-Komplexe eingebauten Nuk-Makromolekülen
• f) Entfernung der Linker-Komponente und der Marker-Komponente von den in die NSK-Primer-Komplexe eingebauten Nuk-Makromolekülen
• g) Waschen der NSK-Primer-Komplexe

gegebenenfalls Wiederholung der Schritte (c) bis (g),Another aspect 51 of the invention relates to a method according to aspects 28 to 35 and 37 to 50, which includes the following steps:

• a) Provision of at least one population of single-stranded nucleic acid chains
• b) hybridization of sequence-specific primers to these nucleic acid chains, wherein NSK primer complexes capable of extension are produced
• c) Incubation of at least one type of nuc-macromolecule according to aspects 1 to 25 together with a type of polymerase according to aspect 31 with the NSK-primer complexes provided in steps a and b under conditions which allow the incorporation of complementary nuc-macromolecules , wherein each type of nuc-macromolecule has a characteristic of their mark.
• d) Removal of unincorporated nuc-macromolecules from the NSK primer complexes
• e) detection of the signals of nuc-macromolecules incorporated into the NSK primer complexes
• f) Removal of the linker moiety and the label component from the nuc-macromolecules incorporated into the NSK primer complexes
• g) washing the NSK primer complexes

optionally repeating steps (c) to (g),

One Another aspect 52 of the invention relates to a method according to aspects 28-40, wherein the nucleic acid chains to a solid phase in random Arrangement are coupled.

• a) zu den auf der Oberfläche gebundenen NSKF-Primer-Komplexen eine Lösung zugibt, die eine oder mehrere Polymerasen und ein bis vier Nuk-Makromoleküle enthält, die eine mit Fluoreszenzfarbstoffen markierte Marker-Komponente haben, wobei die bei gleichzeitiger Verwendung von mindestens zwei Nuk-Makromoleküle jeweils an die Marker-Komponente befindlichen Fluoreszenzfarbstoffe so gewählt sind, dass sich die verwendeten Nuk-Makromoleküle durch Messung unterschiedlicher Fluoreszenzsignale voneinander unterscheiden lassen, wobei die Nuk-Makromoleküle strukturell so modifiziert sind, dass die Polymerase nach Einbau eines solchen Nuk-Makromoleküls in einen wachsenden komplementären Strang nicht in der Lage ist, ein weiteres Nuk-Nakromolekül in denselben Strang einzubauen, wobei die Linker-Komponente mit der Marker-Komponente abspaltbar sind, man
• b) die in Stufe a) erhaltene stationäre Phase unter Bedingungen inkubiert, die zur Verlängerung der komplementären Stränge geeignet sind, wobei die komplementären Stränge jeweils um ein Nuk-Makromolekül verlängert werden, man
• c) die in Stufe b) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung nicht in einen komplementären Strang eingebauter Nuk-Makromoleküle geeignet sind, man
• d) die einzelnen, in komplementäre Stränge eingebauten Nuk-Makromoleküle durch Messen des für den jeweiligen Fluoreszenzfarbstoff charakteristischen Signals detektiert, wobei man gleichzeitig die relative Position der einzelnen Fluoreszenzsignale auf der Reaktionsoberfläche bestimmt, man
• e) zur Erzeugung unmarkierter (NTs oder) NSKFs die Linker-Komponente und die Marker-Komponente von den am komplementären Strang angefügten Nuk-Komponenten abspaltet, man
• f) die in Stufe e) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung der Marker-Komponente geeignet sind, man die Stufen a) bis f) gegebenenfalls mehrfach wiederholt,

wobei man die relative Position einzelner NSKF-Primer-Komplexe auf der Reaktionsoberfläche und die Sequenz dieser NSKFs durch spezifische Zuordnung der in Stufe d) in aufeinanderfolgenden Zyklen an den jeweiligen Positionen detektierten Fluoreszenzsignale zu den Nuk-Makromolekülen bestimmt.Another aspect 53 of the invention relates to a method according to aspects 28 to 41, 52 for the parallel sequence analysis of nucleic acid sequences (nucleic acid chains, NSKs) in which
Generates fragments (NSKFs) of single-stranded NSKs of about 50 to 1000 nucleotides in length, which can be overlapping partial sequences of an overall sequence;
the NSKFs bind in a random array using a single or multiple different primers in the form of NSKF primer complexes on a reaction surface, the density of the surface-primed NSKF primer complexes providing optical detection of the signals from individual integrated nuclides. Macromolecules allows, one
Perform a cyclic assembly reaction of the complementary strand of NSKFs using one or more polymerases by

• a) adding to the surface-bound NSKF-primer complexes a solution containing one or more polymerases and one to four nuc-macromolecules having a fluorescent dye-labeled marker component, wherein the simultaneous use of at least two nucl Macromolecules are each located on the marker component fluorescent dyes are selected so that the nuc-macromolecules used can be distinguished from each other by measuring different fluorescence signals, wherein the nuc-macromolecules are structurally modified so that the polymerase after incorporation of such nuc-macromolecule in a growing complementary strand is not able to incorporate another Nuk nacromolecule in the same strand, the linker component with the marker component are cleavable, one
• b) incubating the stationary phase obtained in step a) under conditions which are suitable for extending the complementary strands, wherein the complementary strands are each extended by one nuc-macromolecule;
• c) washing the stationary phase obtained in step b) under conditions which are suitable for removing nuc-macromolecules not incorporated into a complementary strand;
• d) the individual nucleated macromolecules incorporated into complementary strands are detected by measuring the signal characteristic of the respective fluorescent dye, while simultaneously determining the relative position of the individual fluorescence signals on the reaction surface
• e) to generate unlabelled (NTs or) NSKFs, cleave the linker component and the label moiety from the nucleated components attached to the complementary strand;
• f) washing the stationary phase obtained in step e) under conditions which are suitable for removing the marker component, if necessary repeating steps a) to f) several times,

whereby the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs are determined by specific assignment of the fluorescence signals to the nuc macromolecules detected in step d) in successive cycles at the respective positions.

One Another aspect 54 of the invention relates to a method according to aspect 53, characterized in that the steps a) to f) of the cyclic Construction reaction repeated several times, where in each cycle only each use a kind of nuc macromolecules.

One Another aspect 55 of the invention relates to a method according to aspect 53, characterized in that the steps a) to f) of the cyclic Construction reaction repeated several times, with each cycle in each case uses two different types of nuc-macromolecules.

One Another aspect 56 of the invention relates to a method according to aspect 53, characterized in that the steps a) to f) of the cyclic Construction reaction repeated several times, with each cycle in each case uses four different types of nuc-macromolecules.

One Another aspect 57 of the invention relates to a method according to aspect 53, characterized in that the NSKs variants of a known Reference sequence and are the steps a) to f) of the cyclic synthesis reaction repeated several times, alternating in each cycle two differently labeled types of nuc macromolecules and two Unmarked NTs are used and the overall sequences are compared determined with the reference sequence.

One Another aspect 58 of the invention relates to a method according to Aspects 53 to 57, characterized in that one in the NSKFs in each case introduces a primer binding site (PBS), where in double-stranded NSKs at both complementary single strands each introduces a PBS and wherein the primer binding sites are the same for all NSKFs or have different sequences.

One Another aspect 59 of the invention relates to a method according to Aspects 53 to 57, characterized in that the NSKFs with Primers in a solution under conditions that are favorable for the hybridization of Primers to the primer binding sites (PBSs) of the NSKFs are suitable, where the primers are mutually identical or different sequences and then the formed NSKF primer complexes on the reaction surface binds.

One Another aspect 60 of the invention relates to a method according to Aspects 53 to 57, characterized in that the NSKFs first on the reaction surface immobilized and then with Primers under conditions that bring about hybridization the primer to the primer binding sites (PBSs) of the NSKFs suitable wherein NSKF primer complexes are formed, wherein the primers have mutually identical or different sequences.

One Another aspect 61 of the invention relates to a method according to aspects 53 to 60, in which 10 to 100,000 different sequences populations the installation reaction carried out in parallel

One Another aspect 62 of the invention relates to a method according to aspects 53 to 60, at which 100,000 to 100,000,000 different Sequence populations performed the incorporation reaction in parallel.

One Further aspect 63 of the invention relates to a method according to aspects 28 to 62, wherein the sequences of the nucleic acid chains are determined Another aspect 64 of the invention relates to a method in aspects 28 to 63, wherein the marker component is fluorescently labeled

Another aspect 65 of the invention relates to a method according to aspects 41 to 64, wherein the solid phase is independently selected from the group:
Silicon, glass, ceramics, plastics, gels or their modifications

In further aspect 66 of the invention, particularly preferred macromolecular compounds according to aspect 1, wherein the nuc-component includes the following structures, 3A :
In which:
Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications, where L is the link between the nuc-component and the linker Component is (coupling unit L) and X is the coupling site of the coupling unit L at the base, wherein the coupling site is at pyrimidine bases at the 5-position or 4-position on the pyrimidine ring and at the 7-position of the deazapurines.
R ₂ - is independently selected from the group H, OH, or protected OH group
R ₃ - is independently selected from the group H, OH, NH ₂ ,
R ₄ - is H or OH
R ₅ - is independently selected from the group OH, or a protected OH group, or monophosphate group, or diphosphate group or triphosphate group

Another aspect 67 of the invention relates to macromolecular compounds according to aspect 66, wherein the coupling unit L includes the following structural elements:
R ₆ -NH-R ₇ , R ₆ -OR ₇ , R ₆ -SR ₇ , R ₆ -SS-R ₇ , R ₆ -CO-NH-R ₇ , R ₆ -NH-CO-R ₇ , R ₆ -CO-OR ₇ ,
R ₆ -O-CO-R ₇ , R ₆ -CO-SR ₇ , R ₆ -S-CO-R ₇ , R ₆ -P (O) ₂ -R ₇ , R ₆ -Si-R ₇ , R ₆ - (CH ₂ ) n R ₇ ,
R ₆ - (CH ₂ ) _n -R ₇ , R ₆ -A- (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -BR ₇ ,
R ₆ - (CH = CH-) _n -R ₇ , R ₆ - (A-CH = CH-) _n -R ₇ , R ₆ - (CH = CH-B-) _n -R ₇ ,
R ₆ -A-CH = CH- (CH ₂ -) _n -R ₇ , R ₆ - (- CH = CH-CH ₂ ) _n -BR ₇ ,
R ₆ - (C≡C-) _n -R ₇ , R ₆ - (AC≡C-) _n -R ₇ , R ₆ - (C≡CB-) _n -R ₇ ,
R ₆ -AC≡C- (CH ₂ -) _n -R ₇ , R ₆ - (- C≡C-CH ₂ ) nBR ₇ ,
wherein R ₆ - is the nuc-component, R ₇ - is the linker radical and A and B include the following structural elements: -NH-, -O-, -S-, -SS-, -CO-NH-, -NH- CO, -CO-O-, -O-CO-, -CO-S-, -S-CO-, -P (O) ₂ -, -Si-, - (CH ₂ ) _n -, a photolabile group where n - is 1 to 5

One Further aspect 68 of the invention relates to macromolecular compounds according to aspect 66, wherein the linker component is a water-soluble Polymer includes.

Another aspect 69 of the invention relates to macromolecular compounds according to aspect 68, wherein the linker component includes water-soluble polymers independently selected from the following group:
Polyethylene glycol (PEG), polysaccharides, dextran, polyamides, polypeptides, polyphosphates, polyacetates, poly (alkylene glycols), copolymers of ethylene glycol and propylene glycol, poly (olefinic alcohols), poly (vinyl pyrrolidones), poly (hydroxyalkyl methacrylamides), poly (hydroxyalkyl methacrylates) , Poly (x-hydroxy acids), polyacrylic acid, polyacrylamide, poly (vinyl alcohol).

One Further aspect 70 of the invention relates to macromolecular compounds according to aspects 66 to 69, wherein a linker component is an average Length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 to 100,000 atoms.

One Further aspect 71 of the invention relates to macromolecular compounds according to aspects 66 to 69, wherein a marker component is a signaling, has signal-transmitting, catalytic or affine function

One Another aspect 72 of the invention relates to macromolecular compounds according to aspects 66 to 69, wherein a marker component of a structural marker unit exists

One Another aspect 73 of the invention relates to macromolecular compounds according to aspects 66 to 69, wherein a marker component consists of several structural marker units bound to a core component consists

Another aspect 74 of the invention relates to macromolecular compounds according to aspects 72 or 73, wherein a structural marker unit independently includes one of the following structural elements:
Biotin, hapten, radioactive isotope, rare earth atom, dye, fluorescent dye.

Another aspect 75 of the invention relates to macromolecular compounds according to aspects 72 or 73, wherein a structural marker unit independently includes one of the following elements:
Nanocrystals or their modifications, proteins or their modifications, nucleic acid chains or their modifications, particles or their modifications.

Another aspect 76 of the invention relates to macromolecular compounds according to aspect 75, wherein a structural marker unit includes one of the following proteins:
Enzymes or their conjugates and modifications,
Antibodies or their conjugates and modifications,
Streptavidin or its conjugates and modifications,
Avidin or its conjugates or modifications

One Another aspect 77 of the invention relates to macromolecular compounds according to aspect 75, wherein a structural marker unit is one of following types of nucleic acid chains includes: DNA, RNA, PNA, where the length of nucleic acid chains between 10 and 10,000 nucleotides

One Further aspect 78 of the invention relates to macromolecular compounds according to aspects 73 to 77, wherein the core component of the marker component independently one of the following elements includes: water-soluble polymer from the group of: polyamides (e.g., polypeptides), polyacrylic acid and its derivatives, polyacrylamides and their derivatives, polyvinyl alcohols and their derivatives, nucleic acids and their derivatives, streptavidin or avidin and their derivatives, dendrimers, these elements being linear or branched or interlinked could be

One Another aspect 79 of the invention relates to macromolecular compounds according to aspects 66 to 71, 73 to 78, wherein the connection between covalently multiple structural marker units and the core component or affine occurs.

One Another aspect 80 of the invention relates to macromolecular compounds according to aspects 66 to 73, wherein the connection between a structural Marker unit and the linker is covalent or affine

One Further aspect 81 of the invention relates to macromolecular compounds according to aspects 66 to 71, 73 to 78, wherein the connection between the core component and the linker is covalent or affine

One Further aspect 82 of the invention relates to macromolecular compounds According to aspects 66 to 81, wherein only one Nuk component with a coupled linker is coupled to the marker component

One Another aspect of the invention relates to a macromolecular compound according to aspects 66 to 82, wherein only one nuc-component with a coupled linker is coupled to the marker component, wherein the linker one or more fissile under mild conditions Includes connections.

One Further aspect 84 of the invention relates to a macromolecular compound according to aspects 66 to 82, wherein only one nuc-component with a coupled linker is coupled to the marker component, wherein one or more parts of the nuc macromolecule are so modified that only one Nuk component can be incorporated into a growing strand.

Another aspect 85 of the invention relates to a macromolecular compound according to aspects 66 to 71, 73 to 82, wherein several nuc-components are each coupled via a linker to a marker components

One Another aspect 86 of the invention relates to a macromolecular compound according to aspects 66 to 71, 73 to 82, wherein several nuc-components each over a linker coupled to a marker component, wherein the respective Left one or more compounds cleavable under mild conditions includes.

One Another aspect 87 of the invention relates to a macromolecular compound according to aspects 66 to 71, 73 to 82, wherein several nuc-components each over a linker coupled to a marker component, wherein one or several parts of the nuc macromolecule so are modified that only a nuc-component can be incorporated into a growing nucleic acid chain.

One Another aspect of the invention relates to oligonucleotides or polynucleotides the at least one nuc-macromolecule according to aspects 66 to 87 per a nucleic acid chain lock in.

One Further aspect of the invention relates to oligonucleotides or polynucleotides according to aspect 88, wherein oligo- or polynucleotides are RNA, DNA or PNA are whose length between 5 and 50,000 nucleotides.

Comparison of properties Conventionally modified nucleotides and nuc macromolecules:

substrate properties the conventionally modified nucleotides

Of the Influence of different Linker sizes, the chemical linker composition and the different size and texture of low molecular weight markers on the substrate properties of the nucleotides (Wright, Wright et al., Pharmac.Ther 1990, V. 47, p 447, Klevan US Patent 4828979, Lee. et al. Nucleic Acid Research 1992, V. 20, 2471, J. Brandis Nucleic Acid Research, 1999, V.27, 1912) makes it clear that already minor changes at the nucleotide, linker and marker structure to a strong change the substrate properties of the modified nucleotides can lead.

logically let yourself prove that the Coupling of a significantly larger molecule (e.g. a protein) to a conventionally modified nucleotide Repeal of its substrate properties leads, s. Example 34B.

Out For this reason signal amplifying macromolecules could only be detected enzymatic incorporation of the nucleotides into the nucleic acid chain be coupled with the nucleotide.

In spite of the obvious consideration, a multiple label in the form of a macromolecular marker component to couple to nucleotides, did not yet succeed in this step simple combination between conventional nucleotide structures and a macromolecular marker component while retaining to realize the substrate property of the nucleotides.

The Mass of the conventionally modified nucleotides is substantially in the same range as unmodified nucleotides, and relatively low compared to the mass of proteins, e.g. Streptavidin or polymerases. The increase in the mass of the nucleotide through introduction macromolecular components can cause biochemical and alteration of biochemical physikalalsichen properties of nucleotides.

Overcoming This disadvantage of the prior art was surprisingly achieved by the introduction of a macromolecular linker component between the nuc-component and the macromolecular marker component.

Surprisingly retain the nuc-macromolecules of the invention their Substrate activity despite the massive changes in their properties and can be used by enzymes. For example, polymerases build the nuc-macromolecules of the invention in the growing strand. Terminal deoxy-nucleotidyl transferase (TdT) can Nuc-macromolecules couple to a 3 'end of a nucleic acid (Example 34B, C and Example 35).

It should be obvious to a person skilled in the art that the mass of the Nuk-Ma It forms a multiple of the natural nucleotides and exerts a great influence on the nuc-component.

Applications of nuc macromolecules:

The Coupling a macromolecular marker to a substrate for enzymes, to a nucleotide, offers the possibility a broad use of these nuc-macromolecules in different applications in biotechnology, medicine and science.

According to the invention nuc-macromolecules in processes be used, where they serve as substrates for enzymes.

In an embodiment Nuc-macromolecules are used in the methods of labeling of the invention nucleic acids used. In this case, a incorporation reaction of nuc-macromolecules takes place general rules of the enzymatic extension reaction of nucleic acid chains, ("Molecular Cloning", Maniatis, 3rd ed. 2001).

Of the size Advantage of a macromolecular label per one incorporated nucleotide lies in a much higher signal strength compared to conventional modified nucleotides.

One Another advantage is the big one Distance of the marker from the nucleic acid strand, so that no direct interaction between the marker and the nucleic acid strand occur. This has influence, for example on the fluorescent properties of the markers: the fluorescence of the Marker is not quenched by the nucleobases (purines and pyrimidines).

The Possibility, to position a strong signaling marker on a strand end, e.g. through the coupling through the terminal transferase (see example 35) eliminates the need to be consistent with the strand to label conventionally modified nucleotides, so that native strands of the nucleic acids to disposal stand.

He follows the labeling of a nucleic acid chain for example during the enzymatic synthesis of this chain by the incorporation of nuc-macromolecules, so are no more multistep signal amplification steps anymore necessary. Many of the known signal amplification steps, e.g. Biotin-streptavidin antibody or digoxigenin antibody I antibody II run with only moderate yields from, e.g. Signal amplifications in FISH analyzes. fluctuating and poor yields in the label also cause fluctuations and weakness in the signal, which can lead to misinterpretations. Because of the engagement of nuc macromolecules can the break the marking be eliminated. Through the use of nuc-macromolecules with constant signal quantities can also the fluctuations are largely compensated.

The Labeling of nucleic acids can be used in different procedures. The concrete ones Conditions of nucleic acid preparation, the order of the enzymatic steps and detection processes are dependent on individual from the method in which the mark is used.

Process in the liquid phase

In an embodiment The labeling procedure contains the nucleic acid chains to be labeled in the liquid phase, s. Example 34, 35. Other methods, e.g. PCR, can with Nuc-macromolecules according to the invention are carried out.

The Nucleic acid chains labeled in this way can be used, for example, as probes for the Hybridization to other nucleic acids are used.

Solid phase method

In a further embodiment The labeling procedure contains the nucleic acid chains to be labeled at a fixed phase. Examples of this are microspheres (Spherotech Inc, Streptavidin-polystyrene Particle, 2.17μ), example 34C, and on a flat surface bound nucleic acids.

In particular, the nuc-macromolecules are suitable for analytical methods with incorporation into the nucleic acids coupled to a solid phase, such as, for example, mini-sequencing (Suomalainen A et al., Methods Mol Biol. 2003; 226: 361-6. Liljedahl U et al. Pharmacogenetics. 2003 Jan; 13 (1): 7-17, Olsson C et al. Methods Mol Biol. 2003; 212: 167-76), primer extension (Pirrung MC et al., Bioorg Med Chem Lett., 2001 Sep 17; 11 (18): 2437-40, Cai H, et al., Genomics 2000 Jun 1; 66 (2): 135-43, Kurg A et al., Genet Test. 2000; 4 (1): 1-7., Pastins T et al., Genome Res. 1997 Jun; 7 (6): 606- 14). US pat. 6287766, US pat. 2003148284, US pat. 2003082613, EP 1256632 , WO0194639. Single-Molecular Sequencing (Tcherkassov WO 02088382, Seeger WO 0018956, Kartalov WO 02072892).

Nuc-macromolecules with nuc-component one type (for example dT) preferably carry a specific for them Marker component, so that, for example 4 types of nuc macromolecules (corresponding to the dT, dC, dA and dG bases) used simultaneously and can be distinguished. Other labeling schemes are known in Tcherkassov WO 02088382. Depending on the method, non-labeled, natural nucleotides are also into the reaction mixture along with the nuc macromolecules.

In an embodiment Nuc-macromolecules are used in the labeling process just a single nuc-component in the growing nucleic acid strand allow and multiple installation by modifications to the Nuk component, and / or the linker component and / or the marker component prevented becomes. The further installation can be reversible as well as irreversibly prevented become. In the case of a reversible stop he can in another Process step to be repealed, so that further installation reactions can take place. examples for a reversible blockade of the reaction in applications (Metzker et al. Nucleic acid Research 1994, V.22, p. 4259, Canard et al. Gene, 1994, V. 148, 1, Kwiatkowski US Pat. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642. Tcherkassov WO 02088382).

In another embodiment can further nucleotides incorporated after the incorporation of nuc macromolecules so that even several nuc-macromolecules in a complementary strand can be installed one after the other.

The solid phase, for example, a flat surface or globule (Beads) or a type of multi-vessel arrays (e.g., microtiter plate, Nanotiter plate). This nucleic acids with various methods can be coupled to the solid phase (McGall et al. U.S. Patent 5,411,087, Nikiforov et al. U.S. Patent 5,610,287, Barrett et al. U.S. Patent 5,482,867, Mirzabekov et al. US Patent 5981734, "Microarray biochip technology" 2000 M.Schena Eaton Publishing, "DNA Microarrays "1999 M. Schena Oxford University Press, Rasmussen et al. Analytical Biochemistry v.198, p.138, Allemand et al. Biophysical Journal 1997, v.73, p.2064, Trabesinger et al. Analytical Chemistry 1999, v.71, p.279, Osborne et al. Analytical Chemistry 2000, v.72, 5.3678, Timofeev et al. Nucleic Acid Research (NAR) 1996, v.24 p.3142, Ghosh et al. NAR 1987 v.15 p.5353, Gingeras et al. NAR 1987 v.15 p.5373, Maskos et al. NAR 1992 v.20 p.1679). Preferably, the nucleic acids to be analyzed are provided with a primer ("Molecular Cloning", Maniatis, 3rd ed. 2001).

In an embodiment The process involves incorporation of NT macromolecules into a single population uniform, fixed to the solid phase nucleic acid molecules, wherein the marker component of the nuc macromolecule after installation on the extended Primer remains and is not separated.

In a further embodiment The process involves incorporation of NT macromolecules into a single population uniform, fixed to the solid phase nucleic acid molecules, wherein the marker component or its individual components with or without Linker component of the nuc macromolecule during the incorporation reaction or after the incorporation reaction is separated from the nuc-component.

In a further embodiment The method involves the incorporation of nuc-macromolecules in an enzymatic Reaction in parallel on two or more different populations uniform, fixed to the solid phase nucleic acid molecules, wherein these populations addressable positions on the solid phase with the marker component of the nuc macromolecule after the installation at the extended Primer remains and is not separated.

The addressable positions can for example, have the shape of spots, in the case of a flat surface. at the use of beads as a solid phase to different populations of nucleic acids different globules fixed. When using multi-vial arrays (e.g., microtiter plate, or nanotiter plate) are single nucleic acid populations in individual Fused separately.

In a further embodiment The method involves the incorporation of nuc-macromolecules in an enzymatic Reaction in parallel on two or more different populations uniform, fixed to the solid phase nucleic acid molecules, wherein these populations addressable positions on the solid phase to have. The marker component or its individual components with or without linker component of the nuc-macromolecule is used during the incorporation reaction or separated after the installation reaction from Nuk part.

The addressable positions can for example, have the shape of spots, in the case of a flat surface. at the use of beads as a solid phase to different populations of nucleic acids different globules fixed. When using multi-vessel arrays are single nucleic acid populations separated in individual vessels fixed.

• a) Bereitstellung mindestens einer Population an einzelsträngigen Nukleinsäureketten
• b) Hybridisierung von sequenzspezifischen Primern an diese Nukleinsäureketten, wobei extensionsfähige NSK-Primer-Komplexe entstehen
• c) Inkubation von mindestens einer Art der Nuk-Makromoleküle nach Aspekten 1 bis 25 zusammen mit einer Art der Polymerase nach Aspekt 31 mit den in Schritten a und b bereitgestellten NSK-Primer-Komplexen unter Bedingungen, die den Einbau von komplementären Nuk-Makromolekülen zulassen, wobei jede Art der Nuk-Makromoleküle eine für sie charakteristische Markierung besitzt.
• d) Entfernung der nicht eingebauten Nuk-Makromoleküle von den NSK-Primer-Komplexen
• e) Detektion der Signale von in die NSK-Primer-Komplexe eingebauten Nuk-Makromolekülen
• f) Entfernung der Linker-Komponente und der Marker-Komponente von den in die NSK-Primer-Komplexe eingebauten Nuk-Makromolekülen
• g) Waschen der NSK-Primer-Komplexe

gegebenenfalls Wiederholung der Schritte (c) bis (g),In one embodiment of the method, the following steps are performed:

• a) Provision of at least one population of single-stranded nucleic acid chains
• b) hybridization of sequence-specific primers to these nucleic acid chains, wherein NSK primer complexes capable of extension are produced
• c) Incubation of at least one type of nuc-macromolecule according to aspects 1 to 25 together with a type of polymerase according to aspect 31 with the NSK-primer complexes provided in steps a and b under conditions which allow the incorporation of complementary nuc-macromolecules , wherein each type of nuc-macromolecule has a characteristic of their mark.
• d) Removal of unincorporated nuc-macromolecules from the NSK primer complexes
• e) detection of the signals of nuc-macromolecules incorporated into the NSK primer complexes
• f) Removal of the linker moiety and the label component from the nuc-macromolecules incorporated into the NSK primer complexes
• g) washing the NSK primer complexes

optionally repeating steps (c) to (g),

In a further embodiment of the method, the incorporation reaction of NT macromolecules takes place simultaneously on a population of different nucleic acid molecules fixed on the solid phase, these nucleic acid molecules being bound to the solid phase in a random arrangement (Tcherkassov WO 02088382). In this method, sequences of individual nucleic acid chain molecules are determined. The primer-nucleic acid complexes participating in the enzymatic reaction are fixed at a density that allows the detection of signals from single nuc-macromolecules coupled to a single nucleic acid molecule, wherein the density of the fixed primers or nucleic acids may be substantially higher. For example, the density of the primer nucleic acid complexes participating in the incorporation reaction is from 1 complex to 10 μm ² to 1 complex per 100 μm ² , from 1 complex to 100 μm ² to 1 complex to 1000 μm ² , from 1 complex to 1000 μm ² to 1 complex to 10,000 μm ² .

Examples the fixation of the nucleic acids to the solid phase with a resolution, the analyzes to individual molecules are allowed in WO0157248, US2003064398, US2003013101 and WO02088382 shown. A suitable detection apparatus is in the application WO 03031947 describes.

The Number of individual nucleic acid molecules to be analyzed in parallel for example, between 1000 and 100,000, 10,000 to 1,000,000, 100,000 to 10,000,000 molecules. Where the marker component or its individual components with or without linker component of the nuc macromolecule during the incorporation reaction or is separated from the nuc-component after the incorporation reaction.

• a) zu den auf der Oberfläche gebundenen NSKF-Primer-Komplexen eine Lösung zugibt, die eine oder mehrere Polymerasen und ein bis vier Nuk-Makromoleküle enthält, die eine mit Fluoreszenzfarbstoffen markierte Marker-Komponente haben, wobei die bei gleichzeitiger Verwendung von mindestens zwei Nuk-Makromoleküle jeweils an die Marker-Komponente befindlichen Fluoreszenzfarbstoffe so gewählt sind, dass sich die verwendeten Nuk-Makromoleküle durch Messung unterschiedlicher Fluoreszenzsignale voneinander unterscheiden lassen, wobei die Nuk-Makromoleküle strukturell so modifiziert sind, dass die Polymerase nach Einbau eines solchen Nuk-Makromoleküls in einen wachsenden komplementären Strang nicht in der Lage ist, ein weiteres Nuk-Nakromolekül in denselben Strang einzubauen, wobei die Linker-Komponente mit der Marker-Komponente abspaltbar sind, man
• b) die in Stufe a) erhaltene stationäre Phase unter Bedingungen inkubiert, die zur Verlängerung der komplementären Stränge geeignet sind, wobei die komplementären Stränge jeweils um ein Nuk-Makromolekül verlängert werden, man
• c) die in Stufe b) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung nicht in einen komplementären Strang eingebauter Nuk-Makromoleküle geeignet sind, man
• d) die einzelnen, in komplementäre Stränge eingebauten Nuk-Makromoleküle durch Messen des für den jeweiligen Fluoreszenzfarbstoff charakteristischen Signals detektiert, wobei man gleichzeitig die relative Position der einzelnen Fluoreszenzsignale auf der Reaktionsoberfläche bestimmt, man
• e) zur Erzeugung unmarkierter (NTs oder) NSKFs die Linker-Komponente und die Marker-Komponente von den am komplementären Strang angefügten Nuk-Komponenten abspaltet, man
• f) die in Stufe e) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung der Marker-Komponente geeignet sind, man die Stufen a) bis f) gegebenenfalls mehrfach wiederholt,

wobei man die relative Position einzelner NSKF-Primer-Komplexe auf der Reaktionsoberfläche und die Sequenz dieser NSKFs durch spezifische Zuordnung der in Stufe d) in aufeinanderfolgenden Zyklen an den jeweiligen Positionen detektierten Fluoreszenzsignale zu den Nuk-Makromolekülen bestimmt.This method for parallel sequence analysis of nucleic acid sequences (nucleic acid chains, NSKs) includes the following steps:
Generates fragments (NSKFs) of single-stranded NSKs of about 50 to 1000 nucleotides in length, which can be overlapping partial sequences of an overall sequence;
the NSKFs bind in a random array using a single or multiple different primers in the form of NSKF primer complexes on a reaction surface, the density of the surface-primed NSKF primer complexes providing optical detection of the signals from individual integrated nuclides. Macromolecules allows, one
Perform a cyclic assembly reaction of the complementary strand of NSKFs using one or more polymerases by

• a) adding to the surface bound NSKF primer complexes a solution containing one or more polymerases and one to four nuc macromolecules which label one with fluorescent dyes te marker component, wherein the simultaneous use of at least two nuc-macromolecules each of the marker component fluorescent dyes are selected so that the nuc-macromolecules used can be distinguished from each other by measuring different fluorescence signals, wherein the nuc macromolecules structurally are modified so that the polymerase after incorporation of such nuc-macromolecule in a growing complementary strand is not able to incorporate another Nuk nacromolecule in the same strand, the linker component with the marker component are cleavable, one
• b) incubating the stationary phase obtained in step a) under conditions which are suitable for extending the complementary strands, wherein the complementary strands are each extended by one nuc-macromolecule;
• c) washing the stationary phase obtained in step b) under conditions which are suitable for removing nuc-macromolecules not incorporated into a complementary strand;
• d) the individual nucleated macromolecules incorporated into complementary strands are detected by measuring the signal characteristic of the respective fluorescent dye, while simultaneously determining the relative position of the individual fluorescence signals on the reaction surface
• e) to generate unlabelled (NTs or) NSKFs, cleave the linker component and the label moiety from the nucleated components attached to the complementary strand;
• f) washing the stationary phase obtained in step e) under conditions which are suitable for removing the marker component, if necessary repeating steps a) to f) several times,

whereby the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs are determined by specific assignment of the fluorescence signals to the nuc macromolecules detected in step d) in successive cycles at the respective positions.

To the incorporation of a nuc macromolecule to the 3'-end of the nucleic acid surprisingly retain the now modified nucleic acid chains the ability for the coupling of further nucleotides to the 3'-hydroxyl group by polymerases, s. Example 34C.

This does not mean that only the nuc-component in the nuc-macromolecules but also those with them Nuc-macromolecules modified nucleic acid chains can, with appropriate structure of the nuc-component, e.g. free OH group, accessible for the Enzymes remain and in different areas of biotechnology find an application. Examples of the applications of modified Oligonucleotides are known to those skilled in the art, e.g. in the primer extension reactions or minisequencing reactions.

Choice of enzymes:

nucleotides play a central role in different metabolic processes, for example during storage and transmission genetic information in the cell ("Genes V" B. Lewin, 1994). They are also nucleotides as an energy source the cell known (ATP, UTP), or Signalvemittler (Messenger, GTP) the intracellular Signaling ("Biochemistry and Pathobiochemistry ", G. Löffler, 2003). Because of this, nucleotides and their analogs become therapeutics and diagnostics used.

Have nuc-macromolecules the potential in different areas of biotechnology to find an application The opportunity a coupling of the nucleotides to a macromolecule while maintaining the substrate properties the nucleotides opened many ways too Targeted addressing of the modified nucleotides within of an organism or a cell, so that nuc macromolecules a new fundamental Model for Represent nucleotide prodrugs.

When Enzymes can For example, different types of polymerases used ("DNA Replication", Kornberg, 2nd Ed. 1992), in particular DNA-dependent DNA polymerases, RNA-dependent DNA polymerases, DNA-dependent RNA polymerases and RNA-dependent RNA polymerases. It can used both thermostable and thermolabile polymerases be such as Klenow polymerase or Taq polymerase. Other examples of possible Polymerases will be understood by one of ordinary skill in the literature cited herein. Another example of Enzymes make transferases, such as terminal deoxynucleotidyltransferase ("Molecular Cloning", Maniatis, 3rd ed. 2001). Other enzymes and proteins (for example kinases, Membrane receptors) the nucleotides as substrates), energy source, Cofactors or accepting as messenger substances can be used become.

Enzymes differ in their ability to accept modified nucleotides as substrates. In this application, only DNA polymerases are given as an example. It should be obvious to a specialist In addition, various functional tests must be used to study and apply certain properties of nucleotides. Examples of different test procedures for the labeling of nucleic acids are described in H. Held et al. Nucleic Acid Research 2002, V. 30, p. 3857, M. Metzger et al. Nucleic Acid Research 1994, V. 22, 4259, M. Herrlein et al. Helvetica Chimica Acta 1994, V.77, p. 586, B. Canard et al. PNAS 1995, V. 92, p. 10859 (3'-esters), Canard US 5798210 , J. Hovinen et al. J. Chem. Soc. Perkin 1994, 1994, 211 and also in other patents and publications cited here.

The appropriate combinations between polymerases and modified nucleotides can for the respective Purpose selected accordingly become. examples for the incorporation of nuc macromolecules in the primers are given in Example 34. The examples given are not intended to be limiting the possibility of use of nuc macromolecules, but should give the expert the difference in properties of Nuc-macromolecules represent to the conventional modified NT.

Nuc-macromolecules or whose intermediates can also used in conventional chemical oligonucleotide synthesis For example, in a solid phase synthesis (Giegrich, "Neue photolabile Protecting groups for light-directed oligonucleotide synthesis ", 1997, Beier," New strategies for the construction of RNA and DNA oligonucleotides ", 1996) the nuc-component of nuc-macromolecules corresponding modifications, which allows chemical coupling to the nucleic acid chain, such as in Herrlein, "Synthesis of modified nucleosides, nucleotides and oligonucleotides ", 1993, Gugler," Construction and Application of systems for the simplified chemo-enzymatic synthesis of Oligonucleotide Hybridization Probes ", 1993, Schmidt," New Methods for Synthesis and Isolation long-chain oligonucleotides ", 1991, Bühler, "New photolabile Protecting groups for the oligonucleotide synthesis ", 2000, Bretzger, "Wege to the preparative Oligonucleotide Synthesis "1991, Stengele, "Automated Oligonucleotide Synthesis Using [beta] -eliminable Protecting Groups ", 1991).

Similar to enzymatically synthesized nucleic acid chains (where both oligo- as well as polynucleotides can be used) retain the chemically Oligo or polynucleotides synthesized their ability for coupling additional nucleotides to the 3'-hydroxyl group at. Thus, you can they in different areas of biotechnology, for example be used as a primer.

One Another aspect of the invention is a method for rapid cleaning Nuc-macromolecules directly before their use in the labeling reaction.

method for sequencing single molecules (e.g., Balasubramanian WO 03048387, Tcherkassov WO 02088382, Kartalov WO02072892) require marked Nucleotides in a very clean condition because of impurities a labeled nucleotide preparation cause a sequence error with unlabeled nucleotides can. For this reason, it is important that the labeled nucleotides preferably are free of unlabeled nucleotides. Many nucleotide modifications that used in such processes have one or more under mild conditions cleavable group (Balasubramanian WO 03048387, Tcherkassov WO 02088382)

During the Storage can such nukeotides decay into a part and represent the source for nucleotide analogues without marking, which when incorporated into the nucleic acid to a Sequence error lead would.

This Problem leads to that one Purification can be performed directly before using labeled nuclides got to. A Standard cleaning for modified nucleotides is, for example, an HPLC purification with water-methanol gradient. After such cleaning, the fraction be worked up with modified nucleotides, for example by lyophilization. Such a cleaning is a complicated procedure represents.

Nuc-macromolecules can according to the invention directly before use by ultrafiltration of least contaminants be freed.

It are selected filters with a pore size, the for nucleotides without marker shares are freely passable. Those modified with a macromolecular marker Nucleotides can but do not go through the filter. By such a cleaning In a short time, it is possible to obtain nuk macromolecules in a very clean state to obtain.

Intermediates of nuc-macromolecules and nuc-macromolecules can also be used with one dermolekularen markers are purified in this manner, for example nucleotides, which are described in Examples 36 and 38.

One Another aspect of the invention is the use of Klenow fragment Exominus of DNA polymerase together with the nuc macromolecules in the enzymatic reactions, wherein the SH group of the cysteine of the Klenow fragment Exo minus the DNA polymerase is chemically modified.

These Modification is preferably a covalent modification. Examples for one such modification represents alkylation on the SH group, e.g. with alpha-halogen acetyl derivatives, such as iodoacetamide and its derivatives, iodoacetate and its derivatives, or with N-maleimide derivatives, more selective SH groups Reagents are in "Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993 CRC Press Inc. described. This modification can also be done by a fluorescent dye. activated Fluorescent dyes that react selectively with SH groups are commercial available, e.g. from Molecular Probes Inc.

In a preferred embodiment The invention provides a selective modification of the Klenow fragment Exo-minus of DNA polymerase on SH group of cysteine. example for the Preparation of such a Klenow fragment exo-minus the DNA polymerase is given under Example 39.

In another embodiment can also other modifications to the DNA polymerase are made, such as Couplings to amino groups of the DNA polymerase.

The on the cyst modified Klenow fragment exo minus the DNA polymerase can in one embodiment instead of an unmodified Klenow fragment exo-minus the DNA polymerase together with nuc macromolecules in the enzymatic incorporation reaction be used.

General notes for the syntheses of nuc macromolecules

The Nuc-macromolecules according to the invention can be obtained different ways are synthesized. The chronological order The coupling steps can vary. For example, a first Linker component will be coupled to the Nuk component, then becomes coupled to the marker component. On the other hand, one or more linkers be coupled to the marker component and then the / the Nuc-components.

The Coupling between individual components of nuc-macromolecules can covalent or affine. Both chemical and enzymatic Coupling to the link individual components can be used. Couplings to amino and thiol groups serve as examples of covalent couplings (Jameson, D., et al., Methods in Enzymology 1997, V. 278, 363, "The chemistry of the amino group "S. Patai, 1968, "The chemistry of the thiol group "S. Patai, 1974). Biotin-streptavidin binding or hybridization between complementary Nucleic acid strands or Antigen-antibody interactions provide examples of affine couplings.

The Macromolecular markers often offer a variety of coupling possibilities. A macromolecular marker can have multiple coupling sites for the linker have, for example, multiple binding sites for biotin, as in streptavidin the case is. A macromolecular marker can also contain several amino or thiol groups.

If nucleic acids can be used as macromolecular markers, they can have different sections for coupling other macromolecules. Other macromolecules can be bound to a macromolecular marker be, e.g. Enzymes.

One Nuc-macromolecule can carry macromolecular markers with different detection properties, For example, a nuc-macromolecule can have both multiple dye molecules as well Sites for affinity binding (e.g., by hybridization) to others macromolecules wear.

The Coupling between the nuc-component and the linker components is preferably covalent. Many examples of covalent coupling Nucleotides or their analogs are known (Jameson et al., Method in Enzymology, 1997, v. 278, p. 363-, Held et al. Nucleic acid research, 2002, v. 30 3857, Short US Pat. 6579704, Odedra WO 0192284). there For example, the coupling may be to phosphate, amino, hydroxy or Mercapto groups take place.

Often, the linker component can be built in several steps. For example, in the first step, a short linker having a reactive group is coupled to the nucleotide or nucleoside, eg, propargylamine linker to pyrimidines Hobbs et al. US Patent 5,047,519 or other linkers, eg, Klevan US Pat. 4,828,979, Seela US Pat. 6211158, US pat. 4804748, EP 0286028 , Hanna M. Method in Enzymology 1996, v.274, p.403, Zhu et al. NAR 1994 v.22 p.3418, Jameson et al. Method in Enzymology, 1997, v. 278, p. 363-, Held et al. Nucleic acid research, 2002, v. 30, 3857, Held et al. Nucleosides, nucleotides & nnucleic acids, 2003, v. 22, p. 391, Short US Pat. No. 6579704, Odedra WO 0192284, Herrlein et al. Helvetica Chimica Acta, 1994, V. 77, p. 586, Canard US. Pat. 5798210, Kwiatkowski US Pat. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642, Faulstich et al. DE 4418691 , Dissertation "Synthesis of base-modified nucleoside triphosphates and their enzymatic polymerization to functionalized DNA", Oliver Thum, Bonn 2002. Some compounds can be purchased, for example, from Trilink Biotechnologies, Eurogentec, Jena Bioscience.

These short linkers serve as coupling units L or their parts, and are part of the linker component in the final nuc-macromolecule.

in the second step may be the coupling of the nucleotide or nucleosides with a short linker to the linker polymer. polymers with reactive functional groups can be purchased (Fluka).

To the coupling of the nucleotide to the polymer can now be the marker component be paired as a last step.

It is often advantageous to couple a short linker to a nucleoside, then, if necessary, to convert this modified nucleoside into a nucleoside triphosphate (syntheses of triphosphates can be found, for example, in the following references: Held et al., Nucleosides, nucleotides & nnucleic acids, 2003, v. 22, p. 391, Faulstich et al. DE 4418691 , T. Kovacs, L. Ötvös, Tetrahedron Letters, Vol. 29, 4525-4588 (1988) or dissertation "Synthesis of base-modified nucleoside triphosphates and their enzymatic polymerization to functionalized DNA", Oliver Thum, Bonn 2002).

Further Modifications can be performed on the nucleoside triphosphate analog.

precursors for modified Nucleosides can are for example available from Trilink Biotechnologies (San Diego, CA, USA) or at Chembiotech (Münster, Germany) commercial.

The Coupling between the linker component and the marker component for example, between reactive groups on the linker component and the marker component. Reagents for such couplings are described in "Chemistry of protein conjugation and cross-linking ", Wang, 1993, ISBN 0-8493-5886-8, in detail. The proceedings for handling and coupling of several macromolecules for different Types of macromolecules also shown in the above-mentioned patents. Further examples of couplings to and between the macromolecules are for proteins in "bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2;" Reactive dyes in protein to enzyme technology ", D. Clonis, 1987, ISBN 0-333-34500-2; "Biophysical labeling methods in molecular biology "G. Likhtenshtein, 1993, 1993, ISBN 0-521-43132-8; "Techniques in protein modification "R. Lundblad, 1995, ISBN 0-8493-2606-0; "Chemical Reacts for Protein Modification" R. Lundblad, 1991, ISBN 0-8493-5097-2; For nucleic acids in "Molecular Cloning", J. Sambrook, vol 1-3, 2001, ISBN 0-87969-576-5, for other types of polymers "Macromolecules, Chemical Structure and Synthesis ", Volume 1, 4, H. Elias, 1999, ISBN 3-527-29872-X.

There the marker component usually has many coupling sites, can made further modifications to complete nuc-macromolecules become. For example, excess amino groups be blocked or modified by further modifications.

ever by field of application and reaction conditions under which nuc macromolecules are used can, can different chemical bonding modes between individual parts the macromolecules be beneficial. So are suitable for example in methods with Steps with higher Temperatures, e.g. in hybridization or PCR, nuc macromolecules, the have covalent, thermostable bonds between individual parts.

In the following, some possibilities for the synthesis of nuc macromolecules will be presented by way of example. These are not intended to limit the possible synthesis routes and not to restrict kung of possible nuc-macromolecular structures.

In the following examples nuc-macromolecules are given with polyethylene glycol (PEG) as the linker component. Examples of the coupling of PEG to other molecules are described in "Poly (ethylene glycol): chemistry and biological applications", 1997. In particular, very different reactive groups can be used for the coupling: N-succinimidyl carbonate (US Pat. 5,281,698, US Pat. US 5,468,478 Amine (Buckman et al., Makromol Chem V.182, p.1379 (1981), Zalipsky et al., Eur. Polym JV19, p. 1177 (1983)), succinimidyl propionate and succinimidyl butanoate (Olson et al in Poly (ethylene glycol) Chemistry & Biological Applications, 170-181, Harris & Zalipsky Eds., ACS, Washington, DC, 1997; U.S. Pat No. 5,672,662), succinimidyl succinate (Abuchowski et al., Cancer Biochem., Biophys. v. 7, 5.175 (1984), Joppich et al., Makromol Chem 1v 80, p.1381 (1979), benzotriazole carbonate (US Pat No. 5,650,234), glycidyl ether (Pitha et al., Eur. 94, p.11 (1979), Elling et al., Biotech, Appl. Biochem., V.13, p354 (1991), Oxycarbonylimidazoles (Beauchamp, et al., Anal., Biochem., V.131 25 (1983), Tondelli et al., J. Controlled Release v.1, p.251 (1985)), p-nitrophenyl carbonate (Veronese, et al., Appl. Biochem. Biotech., V.11, P.141 (1985); and Sartore et al., Appl. Biochem Biotech., V.27, p.45 (1991)), aldehydes (Harris et al., J. Polym., Sci., Chem. Ed. 22, p. 341 (1984), US. Pat. 5824784, US. Pat., 5252714), maleimides (Goodson et al., Bio / Technology v.8, p.343 (1990), Romani et al., Chemistry of Peptides and Proteins v.2, p.29 (1984)), and Kogan, Synthetic Comm. v.22, p.2417 (1992)), orthopyridyl disulfides (Woghiren, et al., Bioconj. Chem. v. 4, p.314 (1993)), acrylol (Sawhney et al., Macromolecules, v. 26, P.581 (1993)), vinylsulfones (US Pat. No. 5,900,461). Further examples of couplings of PEG to other molecules are described in Roberts et al. Adv. Drug Deliv. Reviews v. 54, p. 459 (2002), US 2003124086 . US 2003143185 , WO03037385, US6541543, US 2003158333 To find WO 0126692.

Other similar Polymers can in a similar way Art be coupled. Examples of such polymers are others Poly (alkylene glycols), copolymers of ethylene glycol and propylene glycol, Poly (olefinic alcohols), poly (vinylpyrrolidones), poly (hydroxyalkylmethacrylamides), Poly (hydroxyalkyl methacrylates), poly (saccharides), poly (x-hydroxy acids), poly (acrylic acid), poly (vinyl alcohol).

The Purification of the nuc-components of the nuc-macromolecules takes place by conventional means of nucleotide chemistry: for example with silica gel chromatography in a water-ethanol mixture, ion exchange chromatography in a salt gradient and reverse phase chromatography in a water-methanol gradient. For the Nucleotide purification optimized chromatography columns are e.g. offered by Sigma-Aldrich.

The Purification of macromolecular linker components and marker components can be performed by ultrafiltration, gel electrophoresis, gel filtration and dialysis take place, see "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2.

The Mass of nuc macromolecules differs significantly from the mass of nucleotides. For this Reason, it is advantageous in the final purification steps ultrafiltration use. Therefore the nuc macromolecules only an average mass is enriched, ultrafiltration is suitable also as an analytical method for the separation of synthesis products.

to Characterization of nuc macromolecules can be different methods macromolecular chemistry, e.g. UV-Vis spectroscopy, Fluorescence measurement, mass spectroscopy, fractionation, size exclusion chromatography, Ultracentrifugation and electrophoretic techniques, such as IEF, denaturing and non-denaturing gel electrophoresis ("Macromolecules, Chemical Structure and Syntheses", Vol. 1, 4, H. Elias, 1999, ISBN 3-527-29872-X, "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2).

The Measurement of free SH groups in a substance was done with Ellmans Reagent (5,5'-dithioblast (2-nitrobenzoic acid), Riddles et al. Method in enzyme. 1983, V.91, p. 49.

3. Syntheses of modified nucleotides

Separation Methods:

Thin Layer Chromatography, DC:

Analytical: "TLC aluminum foil 20 × 20 cm silica gel 60 F 254" (VWR), coated with fluorescent indicator visualization by means of UV light eluent ethanol-water mixture (70:30) (eluent, LM 1) or ethanol Water mixture (90:10) (LM 2).
Preparative: Silica gel glass plates with collecting layer (VWR). LM 1 or LM 2.

Reverse phase chromatography (RP-chromatography), RP-18:

C-18 Material (Fluka), column volume 10ml, water-methanol gradient. Fractions per 1ml were collected and analyzed with UV-Vis spectrometer. Fractions with similar Spectra were pooled and lyophilized.

HPLC columns with similar Material can be used (Sigma).

Ion exchange chromatography

DEAE-cellulose (VWR), gradient NH ₄ HCO ₃ 20mmol / l - 1mol / l, fractions were collected under UV / Vis control and pooled on the same spectra.

The affinity isolation of nuc macromolecules can be used, for example, when nuc-macromolecules oligonucleotides Component of the marker component are. By hybridizing to a complementary, up A solid phase-fixed nucleic acid can be isolated selectively become.

The Determination of the yields for Dye-labeled products were made on UV-Vis spectrometers.

The completeness The coupling to strepavidin was monitored by a control titration a biotin dye (biotin-4-fluorescein, Sigma) 100 μmol / L in 50 mmol / l borate, pH 8, carried out at RT for 5 min. For a complete cast of biotin binding sites at strepavidin during In the synthesis, no labeling of streptavidin occurs. At a Insufficient reaction occurs binding to SA. analysis with UV-Vis.

Material:

Diamono PEG 10,000 (diamino-polyethylene glycol 10,000, Sigma), dUTP-AA (dUTP-allylamine, Jena-Bioscience, Jena, Germany (hereinafter referred to as Jena-Bioscience)), TTP (thymidine triphosphate), can also be characterized as dTTP , Sigma), 3'-amino-TTP (3'-amino-3'-deoxy-thymidine triphosphate, Trilink Biotechnologies, Inc. San Diego, CA, USA (hereinafter referred to as Trilink)), PDTP (3) (2-pyridinyl-dithio) -propionic acid), PDTP-NHS (3- (2-pyridinyl-dithio) -propionic acid N-hydroxysuccinimidyl ester, Sigma-Aldrich-Fluka, Taufkirchen, Germany (hereinafter referred to as Sigma or Aldrich or Fluka)), Cy3 (Dye, Amerscham Bioscience, Freiburg, Germany, hereinafter referred to as Amersham Bioscience)), Cy3-NHS (Cy3-N-hydroxysuccinimidyl ester, Amerscham Bioscience), MEA (mercaptoethylamine, Sigma), DTT (1,4-dithio-DL-threit, Sigma), CA (cystamine, Sigma), TCEP- (tris (2-carboxyethyl) phosphine, Sigma), DTBP (3,3'-dithio-bis-propionic acid, Fluka ), Biotin-NHS (biotin) N-hydroxysuccinimidyl ester, Sigma). J-Ac (iodoacetate, Sigma), iodoacetamide (Sigma), TEAE (tris (2-aminoethyl) amine, Sigma), maleimido-ES-NHS (maleimido-acetic acid N-hydroxysuccinimidyl ester, Sigma), EDA (ethylene diamine , Sigma), CDI (1,1'-carbonyldiimidazole, Sigma), NHS-PEG-Maleimide, 3,400 Da, Biotin-PEG-NHS, 5,000 Da, mPEG-SPA 5,000 Da, mPEG-SPA 20,000 Da (Nectar Molecular Engineering, formerly Shearwater Corporation, Huntsville, AL, USA, (hereinafter referred to as nectar)), diamine PEG, 6,000 Da (Fluka), 3'-biotin-dT31 an oligonucleotide having a sequence of 31 thymidine monophosphates, with a biotin molecule at 3'-end (MWG-Biotech, Ebersberg near Munich, Germany, (hereinafter referred to as MWG-Biotech)), 3'-SH-oligo-dT30 an oligonucleotide having a sequence of 30 thymidine monophosphates, with a mercapto group at the 3'-end Ende (MWG Biotech, Germany), 3'-amino-oligo-dT31-5'-Cy3 an oligonucleotide having a sequence of 31 thymidine monophosphates, with an Ami coupled via a linker of 6 atoms at the 3 'end no group and one Cy3 dye coupled at the 5 'end (MWG Biotech, Germany), SA (Streptavidin, Roche, Mannheim, Germany), SA-Cy2 (Cy2 dye modified streptavidin, Amersham). QDot (Qdot 605 Streptavidin Conjugate, Quantum Dot, Hayward, Calif., USA (hereinafter referred to as Quantum Dot) Poly-Lysine 1000-2000 (poly-L-lysine hydrobromide 1000-2000 Da, Fluka), poly-lysine 10,000 - 20,000 (poly-L-lysine hydrobromide 10,000-20,000 Da, Fluka).

solvent were, if necessary, Absolutely used (Fluka) or dried by standard methods. For solvent mixtures the specified mixing ratios refer to the used Volumes (v / v).

Synthesis of individual components:

Example 1:

dUTP-AA PDTP, 10A ,

20 mg of dUTP-AA were dissolved in 1 ml of water and the pH was adjusted to 8.5 with NaOH. To the dUTP-AA solution was added PDTP-NHS 60 mg in 0.5 ml of methanol, dropwise with stirring. Reaction was carried out at 40 ° C for 2 hours.
DC control: dUTP-AA-PDTP (in LM 1 Rf 0.45).

The Separation of excess PDTP-NHS and PDTP was preparative Silica gel plates, LM 2. The product, dUTP-AA-PDTP, and dUTP-AA stay on the starting line. The nucleotides were from the plate eluted with water and concentrated. This dUTP analog now has one Disulfide bond, which in a thiol exchange reaction with others Thiols can react and a fissile under mild conditions Represents connection.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 7-deaza-aminopropargyl guanosine triphosphate and 7-deaza-aminopropargyl adenosine triphosphates, 5-amino-propargyl uridine triphosphates may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-deoxyribonuclides can be used, 11 to 14 ,

Example 2:

dCTP-PA PDTP, 10B

The Synthesis was as for dUTP-AA-PDTP, Example 1 described.

Example 3:

dUTP-AA-propionate-SH, 15

To 200μl 40mmol / l solution of dUTP-AA-PDTP was added 1ml TCEP solution 250mmol / l, pH 8, with NaOH, added and stirred for 10 min at RT.

The Separation of the nucleotides from other reagents was preparative Silica gel plates, LM 2nd product, dUTP-AA-propionate-SH, remains on the starting line. The nucleotides were eluted from the plate with water and concentrated.

This dUTP analog carries a reactive SH group which can be easily modified, for example, under Training of disulfide bond.

Example 4:

Biotin-PEG-ethyl-SH, 16

To 200 μl of aqueous CA solution (100 mmol / l), pH 8.5, adjusted with NaOH, 10 mg of biotin-PEG-NHS was added and stirred at 40 ° C for 18 hours. Subsequently, 200 .mu.l TCEP solution (0.5 mol / l), pH 8.0, was added and stirred for a further 10 min at RT. The product was purified by ultrafiltration to 3,000 MWCO (Molecular weight cut off) separated from other reagents. Yield 35%.

The Product carries a reactive SH group that can be easily modified, for example under formation of disulfide bond.

Example 5:

Bis-dithio (Ethyl-PEG-biotin),

To 1 ml of aqueous CA solution (2 mmol / L), pH 8.5 adjusted with NaOH, were 100mg biotin-PEG-NHS added and stirred at RT for 18 hours. The product was through Ultrafiltration to 10,000 MWCO separated from other reagents and lyophilized. Yield 13%.

The Product has a disulfide compound attached to thiol exchange reactions can participate.

Example 6:

MEA-Cy3, 17

To 1ml aqueous CA solution, 200 mmol / l, pH 8.5 adjusted with NaOH, Cy3-NHS was added until the concentration of the Cy3 dye was 10 mmol / l. The reaction was stirred at RT for 10 min. Subsequently was 1 ml of TCEP solution (0.5 mol / L), pH 8.0 adjusted with NaOH, added and more Stirred for 10 min at RT. The product was separated on RP-18 (water-methanol gradient) and concentrated to 0.5 ml, yield 93%, UV-Vis.

The Product carries a reactive SH group that can be easily modified, for example under formation of disulfide bond.

Example 7:

Cy3 TEAE, 18

To 1ml aqueous TEAE solution 300 mmol / l, pH 8.5 adjusted with NaOH, Cy3-NHS was added until the concentration of the Cy3 dye was 5mmol / L. The reaction was stirred at RT for 10 min. The product was separated on RP-18 and concentrated to 0.5 ml, yield 82%, UV-Vis.

The Product has two amino groups that easily react with other reagents can be modified and new functionalities can couple to the dye.

Example 8:

Cy3 TEAE propionate-SH, 19

To 300μl aqueous Cy3-TEAE, 2mmol / L, pH 7.5, was slowly added to 30μl of a freshly prepared methanolic solution of PDTP-NHS, 30mmol / L, added. The course of the reaction was controlled by TLC, LM 1. As reaction products appear on DC substances (LM 1) with Rf. 0.55 (Cy3-TEAE-PDTP) and 0.95 (Cy3-TEAE- (PDTP) 2). After 1 h, the reaction was stopped and the products on DC with LM 1 separated. The substance with Rf. 0.55 was isolated, concentrated and in 200μl Dissolved water. To this solution was 0.1ml TCEP solution (0.5 mol / l), pH 8.0, added and stirred for a further 10 min at RT. The Product, Cy3-TEAE-propionate-SH, was separated on RP-18 (water-methanol gradient) and concentrated to 0.5 ml, yield 26%, UV-Vis.

The Product carries on the one hand, a reactive SH group, which are easily modified can, for example, to form disulfide bond, on the other hand an amino group, which can react with other reagents.

Example 9:

TEAE- (Cy3) 2, 20

To 1ml aqueous TEAE solution 2 mmol / L, Cy3-NHS was added until the concentration of Cy3 dye 4mmol / l was. The reaction was stirred at RT for 10 min. The Product (Rf. 0.45) was on preparation. Silica gel DC with LM 1 separated from other reaction products, with 50mM borate buffer, pH 9, eluted, then collected on RP-18 and subsequently eluted with 50% Et-OH and concentrated to 0.5 ml, yield 22%, UV-Vis.

Example 10 21

Poly-lysine (Cy3) n, n = 10-15, poly-lysine 10,000-20,000

To 1ml aqueous Poly-lysine solution, 1 mmol / L, Cy3-NHS was added until the concentration of Cy3 dye 18mmol / l was. The reaction was stirred at RT for 40 min. The Separation of modified poly-lysine from dye residues took place by ultrafiltration, 3000 MWCO. Determination of the average number Cy3 molecules took place over UV-Vis spectrometer.

Poly-lysine provides an example for a core component to which several marker units, such as dyes can be coupled. In this experiment, the distribution of Cy3 molecules in poly-lysine population was calculated from the known size differences of the poly-lysine molecules and the mean determined number of Cy3 molecules.

Example 11

TEAE- (Cy3) 2-PDTP and TEAE- (Cy3) 2-propionate-SH, 22 :

To 200 μl TEAE- (Cy3) 2, 1 mmol / l were added 10 mg, PDTP-NHS and stirred for 1 hour at RT. Of the Reaction course was controlled by TLC, LM 1. After 1 h was an almost quantitative conversion of TEAE- (Cy3) 2 (Rf. 0.45) to TEAE- (Cy3) 2-PDTP, (Rf. 0.85) detected. The batch was divided into two equal parts.

The Product, TEAE- (Cy3) 2-PDTP, from the first part was separated on RP-18 and lyophilized. Yield 82%, UV Vis.

The Product carries a disulfide bond that is easily involved in a thiol exchange reaction participate and thus be linked to other connections can.

To the second part was 0.1ml TCEP solution (0.5 mol / l), pH 8.0, added to the batch and 10 min at RT touched. The product, TEAE- (Cy3) 2-propionate-SH, was separated on RP-18, Yield 68%, UV Vis.

The Product carries a reactive SH group that can be easily modified, for example under formation of disulfide bond.

Example 12 23

(HS-propionate) m -polylysine- (Cy3) n, (n = 10-15, m 3-9, poly-lysine 10,000-20,000) To 200μl poly-lysine (Cy3) n, 1 mmol / l were added 10 mg, PDTP-NHS and stirred for 1 hour at RT. The Product, (PDTP) m-poly-lysine (Cy3) n, was purified by ultrafiltration separated from the PDTP residues and then dissolved in 100 .mu.l of water. Thereafter, 0.1 ml of TCEP solution (0.5 mol / l), pH 8.0, was added to the batch and stirred at RT for a further 10 min. The Product, (HS-propionate) m-poly-lysine (Cy3) n, was mixed with 3,000 MWCO separated from the low molecular weight components.

The Product carries several reactive SH groups that can be easily modified, for example, below Training of disulfide bond.

Example 13:

TTP-3'-O-propionate-SH, 24A .

The Synthesis of 3'-modified Nucleotides are according to Gottikh et al. Tetrahedron, 1970, v. 26, 4419-, shepherd et al. Method in Enzymology, 1986, v. 126, p. 682-.

DTBP, 210 mg, were dissolved in 1 ml of DMF. To this solution CDI was added 320 mg and stirred for 1 h at RT. Subsequently were 10μl of methanol After a further 10 minutes, 100 μl of this solution, 1 mol / l, were added to 300 μl of aqueous 100 mmol / l solution of TTP, pH 8.5 adjusted with NaOH, added and about 4 h at RT strong touched. The nucleotides were precipitated isolated with ethanol and then dissolved in 300μl of water. Subsequently was 200μl TCEP solution (0.5 mol / l), pH 8.0, added and after 10 min at RT, the repeated precipitation the nucleotides. The separation of the modified nucleotides from the unmodified is not necessary at this stage. yield 13%, UV-Vis.

The Product carries a reactive SH group that can be easily modified, for example under formation of disulfide bond.

Example 14:

TTP-3'-amino-PDTP, 24B

The Synthesis was as for dUTP-AA, Example 1 described: As starting materials were 3'-amino-3'-deoxy-TTP, 100μl, 10mmol / l Solution, pH 8 and PDTP-NHS used. Yield 19%, UV Vis.

The Product carries a disulfide bond that is easily involved in a thiol exchange reaction participate and thus be linked to other connections can.

Other, at the 3 'end with a binding site, such as a short linker, modified nucleotides can be used. Syntheses are shown, for example, in Metzker et al. Nucleic acid Research 1994, V.22, p. 4259, Canard et al. Gene, 1994, V. 148, 1, Hovinen et al. J. Chem. Soc. Perk. Trans. 1994V.1, p. 211, Herrlein et al. Helvetica Chimica Acta, 1994, V. 77, p. 586, Jameson et al. Method in Enzymology, 1997, v. 278, p. 363, Canard US. Pat. 5798210, Kwiatkowski US Pat. No. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642, Faulstich DE 4418691 ,

Further examples for base-modified nucleotides used as nuc-component can, are described in Balasubramanian WO 03048387. Yet more examples for nucleotide analogs are in "nucleotides Analogous, "Scheit, 1980, ISBN 0-471-04854-2, "Nucleosides and Nucleic Acid Chemistry ", Kısakürek 2000, "Anti-HIV Nucleosides" Mitsuya, 1997, "Nucleoside Analogs in cancer therapy ", Cheson, 1997. A person skilled in the art should realize that others too Nucleosides and nucleotides can be used.

Examples of coupling of linker components and marker components on nuc-components

Example 15:

dUTP-AA-SS-MEA-Cy3, 25

To 100μl, 10mmol / l MEA-Cy3 in 50mM borate, pH 9.5, was 50μl 30mmol / l dUTP-AA-PDTP in 50mM borate, pH 9.5 added. After 1 hour, the dUTP-AA-SS-MEA- (Cy3) was purified by thin layer chromatography, LM 1, Rf. 0.6, separated from MEA-Cy3, Rf. 0.9. Subsequently was dUTP-AA-SS-MEA- (Cy3) on RP-18 separated from dUTP-AA-PDTP. yield 67%, UV-Vis.

It a compound was obtained which has a nucleotide functionality and a carries low molecular weight marker functionality.

The By definition, product is a conventional one modified nucleotide: the linker length is less than 30 atoms, the marker component is low molecular weight. It can be representative of conventionally modified nucleotides with only one low molecular weight Markers occur.

These Compound becomes polymerase (e.g., Klenow exo minus polymerase) accepted as substrate, s. Example 34A.

Example 16:

dUTP-AA-SS-TEAE- (Cy3) 2, 26

dUTP-AA-SS-TEAE- (Cy3) 2 became similar how dUTP-AA-SS-MEA- (Cy3), Example 15, was synthesized TEAE- (Cy3) 2-propionate-SH used instead of MEA-Cy3. Exploit 43%, UV-Vis.

It a compound was obtained which has one nucleotide functionality and two low molecular weight marker functionalities.

The By definition, product is a conventional one modified nucleotide: the linker length is less than 30 atoms, the marker component is low molecular weight. It can be representative of Conventionally modified nucleotides with several low molecular weight Markers occur.

These Compound becomes polymerase (e.g., Klenow exo minus polymerase) not accepted as a substrate. The modification of the nucleotide leads to Loss of substrate properties.

Example 17:

dUTP-AA-SS-propionate TEAE-Cy3, 27

dUTP-AA-SS-propionate TEAE-Cy3 became similar how dUTP-AA-SS-MEA-Cy3, Example 15, was synthesized Cy3-TEAE propionate-SH used instead of MEA-Cy3. Yield 37%, UV-Vis.

These Carries connection a nucleotide functionality and a low molecular weight marker functionality. The linker at the base has a free amino group that can be modified.

The By definition, product is a conventional one modified nucleotide: the linker length is less than 30 atoms, the marker component is low molecular weight. The nucleotide is accepted by polymerases as a substrate.

Example 18:

(AA-dUTP-SS-propionate) m -polylysine- (Cy3) n

• Starting materials: dUTP-AA-PDTP
• (HS propionate) m-poly-lysine (Cy3) n, n = 10-15, m 3-9, poly-lysine 10,000-20,000

To 50μl one solution with dUTP-AA-PDTP, 20mmol / L, in 50mM borate buffer, pH 9.0 20μl of (HS-propionate) m-poly-lysine (Cy3) n, about 1mmol / l, in water, and stirred for 18 h at RT. The Product was purified by ultrafiltration, 30,000 MWCO.

It a compound was obtained which has a nucleotide functionality and a carries macromolecular marker functionalities. The product of the reaction is by definition a conventional one modified nucleotide: the linker length is below 30 atoms, the Marker component is macromolecular.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) are not accepted as a substrate. The modification of the nucleotide to the loss of substrate properties.

Example 19:

dUTP-AA-PEG-biotin, 28

To 100 μl of aqueous solution dUTP-AA, 50 mmol / l, pH 8.0, 10 mg of biotin-PEG-NHS were added and stirred at 40 ° C for 18 hours. Subsequently, the unreacted nucleotide was separated by ultrafiltration, 3,000 MWCO, and the product of the reaction, the dUTP-AA-PEG-biotin, several times with water gewa rule.

These Carries connection a nucleotide functionality and a macromolecular linker. Biotin serves as a coupling unit T. To this coupling unit T can macromolecular structures are coupled, e.g. streptavidin without that Substrate properties of these analogs are lost. This nuc macromolecule serves as a substrate for Polymerases.

biotin can also act as a low molecular weight marker unit with signal-mediating Function, which is coupled to the long linker.

The Product of the reaction is by definition an intermediate of a Nuc-macromolecule: the Linker length is significantly larger than 30 atoms and to the coupling unit T more macromolecules can be coupled become.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-propargylamino-dCTP, 7-deaza-aminopropargyl-dGTP, 5-amino-propargyl-dUTP and 7-deaza-aminopropargyl-dATP ( 29 ) can also be modified as stated above. Both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-deoxyribonuclides can be used, 11 to 14 ,

Example 20:

TTP-3'-amino-PEG-biotin,

The Synthesis was similar as for dUTP-AA-PEG-Biotin, Example 19. The starting materials were 3'-amino-3'-deoxy-TTP and biotin-PEG-NHS used. This connection carries a nucleotide functionality and a macromolecular linker and a small molecule marker moiety (Biotin), which has a signal-transmitting function. To the biotin can coupled macromolecular signal-bearing streptavidin molecules become.

The Product of the reaction is by definition an intermediate of a Nuc Macromolecule: the linker length is significantly larger than 30 atoms, the marker component is low molecular weight.

Also other nucleotide analogs having an amino group in the 3'-position may be similar Be paired way.

Example 21:

dCTP-PA-PEG-maleimide,

The Synthesis was as for dUTP-AA-PEG-biotin, Example 19. As starting materials were dCTP-PA and maleimide-PEG-NHS.

These Compound has a nucleotide functionality and a macromolecular Linker. Coupling unit T at this linker is the maleimide group. Maleimide functionality can be macromolecular signal carrying molecules coupled carrying one or more SH groups.

Maleimide group can also act as a low molecular weight marker unit with signal-mediating Function, which is coupled to the long linker.

The Product of the reaction is by definition an intermediate of a Nuc-macromolecule: the Linker length is significantly larger than 30 atoms, the marker component is low molecular weight. At this marker component can macromolecular Structures are coupled without the substrate properties of this Analogues are lost.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-allylamino-dUTP ,, 5-amino-propargyl-dUTP, 7-deaza-aminopropargyl-dGTP and 7-deaza-aminopropargyl-dATP may also be modified as indicated above. Both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-deoxyribonuclides can be used, 11 to 14 ,

Example 22:

dUTP-AA-SS-propionate TEAE- (Cy3) -PEG-biotin, 30

The Synthesis was similar as for dUTP-AA-PEG-biotin, Example 19 described. The starting materials were dUTP-AA-SS-propionate-TEAE-Cy3 and biotin-PEG-NHS. The separation from unreacted dUTP analog was made by ultrafiltration, 3,000 MWCO.

These Carries connection a nucleotide functionality, a dye, a macromolecular linker and a low molecular weight Marker functionality (Biotin), which has a signal-transmitting function. Biotin can too be considered as a coupling unit T of the linker.

The Product of the reaction is by definition an intermediate of a Nuc-macromolecule: the Linker length is significantly larger than 30 atoms, biotin serves as coupling unit T. To this coupling unit T can macromolecular structures can be coupled without the substrate properties these analogs are lost. This nuc macromolecule serves as a substrate for Polymerases.

Of the Dye serves as a sterically demanding group, which is the enzymatic Incorporation of only one nuc macromolecule in the growing strand allowed by the polymerase. The Properties of such nucleotides are detailed in Tcherkassov WO 02088382 describes.

Example 23:

dUTP-AA-SS-PEG-biotin, 31A .

Szu 100μl, 10mmol / l Biotin-PEG-ethyl-SH in 50mM borate, pH 9.5, was 50μl 30mmol / l dUTP-AA-PDTP in 50mM borate, pH 9.5. Reaction was at RT for 18 hours touched. The separation is similar as for the synthesis of dUTP-AA-PEG-biotin, Example 19 described.

These Carries connection a nucleotide functionality and a macromolecular linker. Biotin serves as a coupling unit T. To this coupling unit T can macromolecular structures are coupled, e.g. streptavidin without that Substrate properties of these analogs are lost. This nuc macromolecule serves as a substrate for Polymerases. about Can streptavidin also other macromolecules, For example, enzymes or Nukleinsäurenge be coupled.

The Product of the reaction is by definition an intermediate of a Nuc Macromolecule: the linker length is significantly larger than 30 atoms.

biotin can also be used as signal-mediating low-molecular-weight marker unit be designated.

The Linker component can be used together with the marker component of the Nuk component under mild Conditions are split off. This can be an advantage, for example when sequencing by synthesis (Balasubramanian WO 03048387, Tcherkassov WO 02088382, Quake WO0132930, Kartalov WO02072892) carried out which requires removal of the markers after each detection step is.

Another example of the nuc macromolecules with a cleavable group under mild conditions: dUTP-AA-propionate-S-CO-PEG-biotin ( 31B ).

For this Reaction, the dUTP-AA-PDTP was first purified on RP-HPLC in water-methanol gradient.

To 10μl one 50 mmol / l solution of dUTP-AA-PDTP in water, 20 μl of a 100 mmol / l solution of TCEP, pH 8, added. The reaction was carried out at RT for 10 min. The Product of the reaction, the dUTP-AA-propioate-SH, was from others Reagents on preparative Silica gel plates, LM 2, separated. The product, dUTP-AA-Propionate-SH, stays on the starting line. It was eluted from the plate with water, concentrated and in 50 ul 50 mmol / l borate buffer, pH 8, dissolved.

To this solution was added 50 μl of freshly added 2% aqueous solution of biotin-PEG-NHS. The reaction was carried out at RT for 30 min. Subsequently, the product of the reaction, the dUTP-AA-propionate-S-CO-PEG-biotin, separated from low molecular weight reaction components by ultrafiltration with MWCO 3000, washed five times with 0.5 ml of water and dissolved in a volume of 50 μl.

The thus, the dUTP-AA-propionate-S-CO-PEG-biotin was obtained may be of DNA polymerases, for example Klenow fragment Exo minus or Taq polymerase, incorporated into the growing strand of nucleic acids become.

At The biotin can be over that Straptavidin further marker components can be coupled.

The the dUTP-AA-propionate-S-CO-PEG-biotin contains one under mild conditions fissile group, so that the Linker with the dye can be cleaved from the nucleotide. This is for example in methods of sequencing by the Synthesis of Special Interest (Balasubramanian WO 03048387, Tcherkassov WO 02088382, Quake WO0132930, Kartalov WO02072892).

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-propargylamino-dCTP, 5-amino-propargyl-dUTP, 7-deaza-aminopropargyl-dGTP, and 7-deaza-aminopropargyl-dATP may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-deoxyribonuclides can be used, 11 to 14 ,

Example 24:

TTP-O-propionate-SS-PEG-biotin,

to 100μl solution of TTP-O-propionate-SH, 10mmol / L, in 50mM borate buffer, pH 9.5 100μl one solution of bis-dithio (ethyl-PEG-biotin), 0.5mmol / L, in water and stirred at RT for 24 hours. The Purification was done by ultrafiltration with 3,000 MWCO similar to in example 19.

Examples for the Coupling of amino groups to the phosphate radicals of the nucleotides are in D. Jameson et al. Methods in Enzymology 1997, v. 278, 363. To this amino group, a linker component can be coupled. examples for Couplings of linkers to phosphate groups of nucleotides are in U.S. Patent 5,981,507. To such a linker can more macromolecular linker coupled to a low molecular weight Marker or a low molecular weight coupling unit or a markomolecular Bear markers. In an embodiment is added markomolekularer linker to the phosphate groups, the at the 5'-position the ribose are coupled. The coupling is preferably on the gamma-phosphate group of the nucleotides, wherein both ribonucleotides as well as 2'-deoxyribonucleotides as well as 2 ', 3'-dideoxynucleotides can be used.

In another embodiment follows the coupling of the macromolecular linker to the 3'-phosphate group a 5'-nucleoside triphosphate. Synthesis of such a derivative is shown in WO 91/06678.

Couplings to marker components

Example 25

(DUTP-16-biotin) 4-SA,

To 200μl one solution of biotin-16-dUTP 200μmol / l in 50 mM Tris-HCl, pH 8.0, 200 μl of a streptavidin solution, 1 mg / ml, in 50 mM Tris-HCl, pH 8.0. After 1 hour at RT was was the (dUTP-16-biotin) 4-SA from unreacted biotin-16-dUTP separated by ultrafiltration, 50,000 MWCO.

It a compound was obtained which has a nucleotide functionality and a carries macromolecular marker functionalities.

The Product of the reaction is by definition a conventionally modified Nucleotide: the linker length is less than 30 atoms, the marker component is macromolecular. It can be representative of conventional considered modified nucleotides with a macromolecular marker become.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) are not accepted as a substrate. The modification leads to Loss of substrate properties, s. Example 34B.

properties biotin-streptavidin binding are described, for example, in Gonzalez et al. journal Biolog. Chem. 1997, v. 272, p 11288 described.

Example 26:

(DUTP-16-biotin) 4-SA-Cy2,

The Coupling of dUTP-16-biotin to SA-Cy2 was as for (dUTP-16-biotin) 4-SA described carried out.

The Connection serves as an equivalent to compound of Example 25, wherein for visualization streptavidin fluorescently labeled.

Example 27:

dCTP-PA-SS-oligo-dT30,

The Synthesis was as described for dUTP-AA-SS-MEA-Cy3. To dCTP-PA-PDTP, 100μl, 20mmol / l, became oligo-dT30-3'-SH (MWG Biotech) added, final concentration 200μmol / l, and 18 h at RT, pH 9, stirred. The purification was carried out by ultrafiltration with 3,000 MWCO.

The Product of the reaction is by definition a conventionally modified Nucleotide analog: the linker length is less than 30 atoms, the marker component is macromolecular.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) are not accepted as a substrate. The modification of the nucleotide portion leads to abolish the substrate properties.

Example 28:

(dUTP-AA-PEG-biotin) 4-SA-Cy2 and (dUTP-AA-PEG-biotin) 4-SA, 32

The Coupling of dUTP-AA-PEG-biotin to SA-Cy2 or to SA was described as for (dUTP-16-biotin) 4-SA carried out. To 200μl 1 μg / μl streptavidin were 10μl a solution of dUTP-AA-PEG-biotin, ca. 1 mmol / l, and stirred at RT for 1 h. After that the product was not ultrafiltered by 50,000 MWCO of that coupled dUTP-AA-PEG-biotin separated and the product 2 times washed with water.

One Part of the recovered (dUTP-AA-PEG-biotin) 4-SA-Cy2 was digested with Cy3-NHS modified: 50 μl (dUTP-AA-PEG-biotin) 4-SA-Cy2 were dissolved in 50 mmol / l borate, pH 8.5, up to the concentration of 1.4 μg / μl, and then with Cy3-NHS spiked. Final concentration of Cy3 was 10 mmol / l. The Reaction was carried out at RT for 1 h. The product, (dUTP-AA-PEG-biotin) 4-SA-Cy2 / Cy3, was separated by ultrafiltration with 30,000 MWCO.

Thereby became a nuc macromolecule prepared that has very few free amino groups on the marker part.

It a compound was obtained which has a nucleotide functionality, a long macromolecular linker and a macromolecular marker component wearing. The product of the reaction is by definition a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. It can be representative considered for nuc macromolecules become.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) as substrate s. example 34.35.

Also other compounds that have a long linker and carry a biotin molecule, Examples 20, 22, 23, 24 may be similar in FIG the synthesis can be used.

Example 29:

(dUTP-AA-PEG-Biotin) 4-SA-Alkaline Phosphatase, 33 , and (dUTP-AA-PEG-biotin) 4-SA-QDot, 34 .

The Coupling of dUTP-AA-PEG-biotin to SA-AP and QDot, respectively, has been described as for (dUTP-16-biotin) 4-SA carried out.

in the Traps of QDot become nuc-linker parts on the surface of the QDots arranged. There were obtained compounds which have a nucleotide functionality, a long macromolecular linker and a macromolecular marker component carries with an enzyme or Q-Dots.

The The product of the reaction is by definition a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. This connection is derived from polymerases (e.g., Klenow exo minus polymerase and terminal Transferase) as a substrate.

Also other compounds that have a long linker and carry a biotin molecule, Examples 20, 22, 23, 24 may be similar in FIG the synthesis can be used.

Example 30:

(DUTP-AA-PEG-biotin) 2 (dT31-TEG-biotin) 2-SA-Cy2 ,, 35A

The coupling of dUTP-AA-PEG-biotin to SA-Cy2 was performed as described for (dUTP-16-biotin) 4-SA:
To 100 μl of a streptavidin-Cy2 solution, 20 μmol / l (1.2 mg / ml) in Tris-HCl, 50 mmol / l, pH 8, was added 80 μl 80 μmol / l dT31-3'-TEG-biotin (MWG Biotech) and Incubated at RT for 10 min. (TEG is a short linker between biotin and dT31). Thereafter, 100 μl of a solution of dUTP-AA-PEG-biotin 50 μmol / l in 50 mM Tris-HCl, pH 8.0, was added. After 10 min at RT, (dUTP-AA-PEG-biotin) 2- (dT31-TEG-biotin) 2-SA-Cy2 was purified by ultrafiltration, 50,000 MWCO.

The substance carries a nucleoside triphosphate functionality and a macromolecular marker functionalities (oligo-dT31). The oligo-dT31 consists of nucleoside monophosphates, which, however, do not participate in the enzymatic reaction and have only one signal-transmitting function. To such an oligonucleotide complementary nucleic acids can be hybridized, which have a signaling function ( 35B ). (general rules for the hybridization of nucleic acids are known in the art, Anderson "Nucleic Acid Hybridization", 1999).

The The product of the reaction is by definition a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. This connection is derived from polymerases (e.g., Klenow exo minus polymerase and terminal Transferase) as a substrate.

For example, this derivative can be coupled to poly-dA or poly-A (eg, 260NT average length, Amersham Bioscience) by hybridization. Both a single and several (dUTP-AA-PEG-biotin) 2- (dT31-biotin) 2-SA-Cy2 molecules can be coupled to a poly-dA molecule, s 5 , The ratio is determined by the concentration ratios. Other oligonucleotides, for example labeled with dyes, can be coupled together with (dUTP-AA-PEG-biotin) 2- (dT31-biotin) 2-SA-Cy2 to the same strand of poly-dA or poly-A, the ratios between individual molecules are variable. This makes it possible to produce a polyfunctional nuc macromolecule. The great advantage of such a nuc-macromolecule consists in a readily cleavable macromolecular labeling: the labeled oligonucleotides hybridized to the poly-dA or poly-A strands can be removed by denaturation. By choosing the length of the labeled oligonucleotides, the Tm of these oligonucleotides can be adapted for the respective requirements of the reversible label. The rules for Tm calculation are known in the art ("Molecular Cloning", J. Sambrook, vol. 1-3, 2001). For example, dT ₂₅ oligonucleotides labeled with a Cy3 molecule can be coupled to poly-dA. By using RNA, eg poly-A, to bind several (dUTP-AA-PEG-biotin) 2- (dT ₃₁ -biotin) 2-SA molecules, the cleavage can be carried out by an RNase.

Since streptavidin has 4 binding sites for biotin, a mixture of nuc macromolecules is formed in which 4 binding sites are differently occupied. This mixture can ge by different means be separated. One possibility is to isolate nuc macromolecules bearing at least one oligo-dT31, for example by absorption on an anion exchanger (eg a DEAE-cellulose column). Gel electrophoresis is suitable for the separation of individual derivatives.

Longer nucleic acid chains bearing a biotin molecule, for example poly-dA-biotin, prepared, for example, by a terminal coupling of ddUTP-18-biotin by a TdT-dependent reaction ("Molecular Cloning", J. Sambrook, Volume 1). 3, 2001) can be similarly coupled to the streptavidin to form molecules having an average composition of (dUTP-AA-PEG-biotin) _N- (nucleic acid chain-biotin) _M -SA. Both single-stranded and double-stranded nucleic acid chains can be coupled. The length of the coupled nucleic acid chains may be between 10 and 100, 100 and 1000 nucleotides.

The hybridized oligonucleotides carrying a dye may be attached The poly-dA strand can also be covalently bound by cross-linkers.

Also other compounds that have a long linker and carry a biotin molecule, Examples 20, 22, 23, 24 may be similar in FIG the synthesis can be used.

Example 31:

(dUTP-AA-SS-PEG-biotin) 4-SA and (dUTP-AA-SS-PEG-biotin) 4-SA-Cy2, 36

The Coupling of dUTP-AA-SS-PEG-biotin to SA or to SA-Cy2 was like for (dUTP-AA-PEG-biotin) 4-SA described carried out. The starting materials used were streptavidin and dUTP-AA-SS-PEG-biotin.

It a compound was obtained which has a nucleotide functionality, a long macromolecular linker and a macromolecular marker component wearing. The linker component and the marker component can be of the nuk component are cleaved under mild conditions.

The The product of the reaction is by definition a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. It can be representative considered for nuc macromolecules which are a fissile compound in mild conditions bear the linker component.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) as substrate s. example 34th

Example 32:

dCTP-PA-PEG-maleimide S-oligo-dT30, 37A

To 100μl one solution with dCTP-PA-PEG-maleimide, 5mmol / L, in 50mM borate buffer, pH 9.5, was 100μl a solution of 3'-SH-oligo-dT30, 200μmol / l, added in water and stirred for 48 hrs. At RT. The product was using preparative Gel electrophoresis 12% polyacrylamide gel, cleaned.

The substance carries a nucleoside triphosphate functionality and a macromolecular marker functionalities (oligo-dT30). The oligo-dT30 consists of nucleotides, which, however, do not participate in the enzymatic reaction and have only one signal-transmitting function. To such an oligonucleotide complementary nucleic acids can be hybridized, which have a signaling function ( 37B ). (general rules for the hybridization of nucleic acids are known in the art, Anderson "Nucleic Acid Hybridization", 1999.) The nucleotide is by definition a nuc-macromolecule: the linker length is significantly greater than 30 atoms, the marker component is macromolecular.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) as a substrate.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-allylamino-dUTP, 5-amino-propargyl-dUTP, 7-deaza-aminopropargyl-dGTP, and 7-deaza-aminopropargyl-dATP may also be modified as noted above. It can both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-Deoxyribonukletide ver to be turned, 11 to 14 ,

By adding poly-dA or poly-A, several dCTP-PA-PEG-maleimide-S-oligo-dT30, eg 10-20, can be coupled to a nuc macromolecule. This results in a nuc macromolecule with linearly arranged nuc-linker components ( 37C ).

Example 33

(DCTP-PA-PEG-maleimide-S) n-Poly-lysine (Cy3) m,

• Starting materials: dCTP-PA-PEG-maleimide
• (HS propionate) m-poly-lysine (Cy3) n, n = 10-15, m 3-9, poly-lysine 10,000-20,000

To 100μl one solution with dCTP-PA-PEG-maleimide, 5mmol / L, in 50mM borate buffer, pH 9.5, was 20μl a solution of (HS-propionate) m-poly-lysine (Cy3) n, ca. 1mmol / L, in water and 18 hours at 40 ° C touched. The product was purified by ultrafiltration, 30,000 MWCO.

It a compound was obtained which has a nucleotide functionality, a long macromolecular linker and a macromolecular marker component wearing. Pro Nuc Macromolecule Several Nuk components are coupled. The product of the reaction is by definition a nuc macromolecule: the linker length clearly greater than 30 atoms, the marker component is macromolecular.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) as a substrate.

Further Combinations of nuc-components, linker components and marker components are the Skilled person.

Comparison of substrate properties some representatives of nuc macromolecules with conventionally modified nucleotides.

substrate properties the nuc macromolecules across from Polymerases and terminal deoxy-nucleotidyl transferase (TdT) were with the properties of the conventionally modified nucleotides compared in a labeling reaction. General principles of Labeling reactions can be found in "Molecular Cloning", J. Sambrook, vol. 1-3, 2001, ISBN 0-87969-576-5.

Example 34: Substrate Properties of nuc macromolecules or conventionally modified nucleotides towards polymerases

This Example is not intended to be limiting the possible Marking reactions serve, but only differences in illustrate the substrate properties.

In The reactions were both self-synthesized and commercial available modified nucleotides dUTP-Cy3 (Amersham) and dUTP-16-biotin (Roche) used. Unmodified dNTP (dATP, dGTP, dTTP, dCTP) were added acquired from Roth.

When Matrices served both short oligonucleotides and poly-dA. Primers and oligonucleotides were synthesized by MWG-Biotech.

Reactions were performed in 20mmol / L Tris buffer, pH 8.5, 5mmol / L MgCl ₂ , 10% glycerol. The concentrations of the primers were 1 μmol / l, those of the oligonucleotides 1 μmol / l and the concentration of the poly-dA, 0.1 μg / μl (for the concentration ratios of the solid phase see below). The polymerase used was Klenow Exo minus 1 unit / 100 μl (Amersham). The concentrations of nucleotides were 20 μmol / L for conventionally modified nucleotides and 5 μmol / L for nuc macromolecules. Unmodified nucleotides were used in concentrations of 50 μmol / l.

At first were Primer hybridized to the respective template: The reaction mixture without polymerase was at 75 ° C heated and over 5 min at 37 ° C cooled. Thereafter, the polymerase was added. All reactions were over 1 h at 37 ° C carried out. The reactions were monitored by addition of EDTA (final concentration of 10mmol / l) stopped.

To some approaches was after stopping the reaction streptavidin, up to final concentration of 1 mg / ml, and the mixture was incubated for a further 10 min at 37 ° C. Thereby can already incorporated nucleotides carrying a biotin with streptavidin react and thus connect streptavidin and oligonucleotide. These Experiments are suitable as a control for the running properties of modified primers.

To labeled accordingly was mercaptoethanol (to 20mmol / l final concentration) was added and the respective batch was 10 min at 37 ° C incubated. Mercaptoethanol was added during some attacks during other approaches also added after the reaction.

The Analysis of the reaction was carried out by means of denaturing gel electrophoresis, 20% polyacrylamide gel, 50 mmol / l Tris-HCl, pH 8.7, as described in "Gel electrophoresis of nucleic acids ", Ed. D. Rickwood, 1990. For denaturing the samples instead of 7M urea increased gel-electrophoresis temperature used (60 ° C). Electrophoresis was carried out in Biorad gel chambers (Protean 3), 200V, about 1h. The visualization was carried out on a UV-Vis gel documentation system (Biorad).

Example 34A, 38

Presentation of the installation and cleavage of a conventionally modified nucleotide (dUTP-AA-SS-MEA-Cy3)

sequences:

Legend:

Lanes 1-6:

1)

only primer T7-20-Cy3 + Oligo 1

2)

Primer T7-20-Cy3 + Oligo 1 + dCTP-Cy3 + dATP + dGTP + polymerase

3)

Primer T7-20-Cy3 + Oligo 1 + dCTP-Cy3 + dATP + dGTP + dTTP + polymerase

4)

Primer T7-20-Cy3 + Oligo 1 + dUTP-AA-SS-MEA-Cy3 + polymerase

5)

after 1 hour was added to an aliquot of batch 4 mercaptoethanol and more Incubated for 10 min, there is a cleavage of the label

6)

after 10 min was added to the aliquot of batch 5 dATP, dGTP and incubated at 37 ° C for 30 min

As can be seen, dUTP-AA-SS-MEA-Cy3 is incorporated by the polymerase (lane 4). The dye can be cleaved from the primer (lane 5) (one sees a shift of the band, which is due to the smaller size of oligonucleotide). Subsequently, further nucleotides can be incorporated (lane 6).

A similarly carried out reaction with dUTP-AA-SS-TEAE- (Cy3) 2 resulted not for incorporation of the nucleotide analog into the primer.

This Example shows that even minor changes in the analog structure, e.g. Doubling the number of dyes, which are coupled to a nucleotide, to change in substrate properties the nucleotides lead can.

Example 34B 39

Comparison of substrate properties a conventionally modified nucleotide with a macromolecular marker and a nuc macromolecule

sequences:

Legend:

Tracks 1-9:

1)

Head: T-7-20-Cy3, dT35-Cy3, dT40-Cy3, dT50-Cy3

2)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase

3)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + poly-dA

4)

(DUTP-16-biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase

5)

(DUTP-16-biotin) 4-SA + dT35-Cy3 + poly-dA

6)

(DUTP-16-biotin) 4-SA-Cy3 + dT35-Cy3 + poly-dA

• In the control approach, lanes 7-9:

  7)

  dUTP-16-biotin + dT35-Cy3 + poly-dA + polymerase, incubation 1 hr. 37 ° C then + EDTA to 10mmol / L final concentration, then + streptavidin 10 min 37 ° C

  8th)

  dUTP-16-biotin + dT35-Cy3 + poly-dA + polymerase, incubation 1 hr. 37 ° C then + EDTA to 10mmol / L final concentration,

  9)

  Ladder: T-7-20-Cy3, dT35-Cy3, dT40-Cy3, dT50-Cy3

One Nuc-macromolecule (dUTP-AA-SS-PEG-biotin) 4-SA, is incorporated into the primer (lane 2). The labeled primer has one after incorporation of a nuc macromolecule strongly changed electrophoretic mobility. Has only the presence of nuc macromolecules no influence on the primer (lane 3).

One conventionally modified nucleotide, (dUTP-16-biotin) 4-SA, with a macromolecular marker is not incorporated into the primer (Lane 4). Despite the presence of the polymerase in reaction 4 (lane 4) no differences between lane 4 and lane 5 can be detected.

track Figure 6 shows the position of the conventionally modified nucleotide with a macromolecular marker, (dUTP-16-biotin) 4-SA-Cy3, the upper band, and the position of the labeled primer (the lower Band).

track Figure 7 shows the result of incorporation of dUTP-16 biotin with a subsequent one Reaction with streptavidin: the biotin-mimicked primer react with streptavidin and change their running properties. The unmodified primers retain their electrophoretic properties at.

track 8 shows the result of the incorporation reaction of a conventionally modified one Nucleotides, dUTP-16-biotin. You see a common primer band, which is due to the incorporation of dUTP-16-biotin into the primer. The extension Primer is restricted, because dUTP-16-biotin are not incorporated indefinitely one after the other On average, about 3 dUTP analogs are incorporated, so that the length of Average primer rises to 38 NT. As expected, the leads Incorporation of conventionally modified nucleotides with a low molecular weight Marker does not cause a strong change in the electrophoretic mobility the primer.

In properties of the nuc macromolecules (dUTP-AA-SS-PEG-biotin) 4-SA, compared with those of conventionally modified nucleotides. It is clear that the Coupling of a macromolecular marker to a commercially acquired dUTP-16-biotin to complete Loss of substrate properties of the nucleotides leads. traces However, FIGS. 7 and 8 show that the Polymerase is well able to dUTP-16-biotin without macromolecular Insert markers into the primer. The coupling of streptavidin the biotin after the incorporation reaction leads to said changes in primer properties.

in the Contrary to this Nuc-macromolecules (dUTP-AA-SS-PEG-biotin) 4-SA in the primer without difficulty the polymerases are incorporated. The appearance of several gangs in the gel lead the Inventor of a multiple incorporation of nuc macromolecules in the Primer (3 bands) back.

Example 34C, 40 :

Comparison of substrate properties of nuc macromolecules, Incorporation reaction in the solution and at a fixed phase

sequences:

Preparation of streptavidin-polystyrene Particle (solid phase).

Three approaches were prepared in the same way.

0.5 ml of the solution with beads (in the manufacturer buffer) was briefly centrifuged and pellet pellets were resuspended in 100 μl of incorporation buffer (20 mmol / l Tris, pH 8.5, 5 mmol / l MgCl 2). Then 100 μl of oligo-dA50 3'-TEG-biotin conc. 50 μmol / l were added and the mixture was stirred at RT for 1 h. In this time, the bind Oligo-dA molecules to the beads. Subsequently, beads were briefly centrifuged off and washed three times in the built-in buffer. Final volume of the solid phase amounted to 100 μl. This amount of oligo-dA50 solid phase can hybridize primer dT-35-Cy3 in 2 μmol / L.

The Hybridization of primer dT-35-Cy3 was carried out at 40 ° C for 10 min, with following Cooling to RT within 10 min. All other steps became the same in all approaches carried out.

Legend:

Tracks 1-10:

1)

Conductor: dT35-Cy3, dT40-Cy3,

Reactions in the liquid phase:

2)

(DUTP-AA-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase

3)

(DUTP-AA-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA

4)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase, incubation for 1 h at 37 ° C, then + EDTA

5)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase, incubation for 1 h at 37 ° C, then + EDTA, subsequently + Mercaptoethanol up to 200mmol / L (final concentration), for 30 min.

6)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + poly-dA + EDTA

Reactions at the solid phase:

7)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + oligo-dA50 solid phase + polymerase, incubation for 1 h 37 ° C, then + EDTA

8th)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + oligo-dA50 solid phase + polymerase, incubation for 1 h 37 ° C, then + EDTA, then + Mercaptoethanol up to 200mmol / L (final concentration), for 30 min.

9)

(AA-dUTP-SS-PEG-Biotin) 4-SA + dT35-Cy3 + oligo-dA50 solid phase, before electrophoresis + EDTA

10)

Conductor: dT35-Cy3, dT40-Cy3,

you clearly sees the result of the incorporation reaction of nuc macromolecules in traces 2,4,5,7,8. Both in the solution, as well as running on the solid phase the enzymatic labeling reaction is good.

To Add mercaptoethanol to EDTA stopped reactions the cleavage of linker components with the bound streptavidin from the primers. This leads to restore the electrophoretic properties of the primers. The shift of the primer bands in lanes 5 and 8 is by multiple Incorporation of nuc macromolecules to justify in the primer. Indeed, After cleavage, the primer bands are at the level of dT40-Cy3 (see ladder). This means that until to 5 nuc-macromolecules while the reaction were incorporated into the primer.

Example 35 41 :

Substrate Properties the nuc macromolecules and conventionally modified nucleotides versus terminal transferase (TdT).

The reaction was carried out according to the kit manufacturer (Roche): to each 50 .mu.l volume were added: 10 .mu.l 5x reaction buffer, 1 .mu.l TdT (25Units), 5 .mu.l 25mmol / l CoCl ₂ . The primer and nucleotide concentrations were as in the polymerase reactions. The reaction was carried out for 2 h at 37 ° C.
Primer: primer dT ₃₅ -5'-Cy3 (dT35-Cy3), primer dT ₃₅ (dT35)

Legend:

1)

(DUTP-AA-PEG-biotin) 4-SA + dT35-Cy3 + TdT

2)

(DUTP-AA-PEG-biotin) 4-SA + dT35-Cy3

3)

dUTP-Cy3 (Amersham) + dT35 + TdT

4)

dUTP-Cy3 (Amersham) + dT35

5)

dUTP-16-biotin (Roche) + dT35-Cy3 + TdT;

6)

Approach 5; after this Stop + streptavidin

7)

Streptavidin-Cy2

8th)

(DUTP-16-biotin) 4-SA + dT35-Cy3 + TdT

9)

(DUTP-16-biotin) 4-SA + dT35-Cy3

10)

(DUTP-16-biotin) 4-SA-Cy2

In On track 1 you can clearly see 2 bands corresponding to the band in the middle the dT35-Cy3, the upper band corresponds to the reaction product: Nuk macromolecule was installed in dT35 by TdT. Lane 2 serves as a negative control. In lane 3 you can see the result of marking the dT35 with the conventional nucleotide dUTP-Cy3, Lane 4 is the nagative control. In track 5, the result is the Coupling of dUTP-16-biotin to the dT35-Cy3 difficult to detect. In track 6, however, you can see a shabby band in the upper area, the the result of the reaction of dUTP-16-biotin modified primer with Streptavidin corresponds. Lane 7 shows the position of the modified Streptavidin. In lanes 8 and 9 you only see one band in the middle of the gel corresponding to the dT35-Cy3 is in the upper gel region no gang to be seen, which clearly indicates that conventional modified nucleotides with a macromolecular marker of TdT not installed. Lane 10 shows the position of (dUTP-16-biotin) 4-SA-Cy2 in the gel.

Example 36:

dUTP-AA-SS-PEG-Cy3, 42A

First, SH-PEG-Cy3 was prepared. To 200 μl of a 10 mmol / l solution of diamine PEG (6kDa, Fluka) in 50 mmol / l borate buffer, pH 8, Cy3-NHS (Amersham-Bioscience) was added to a final concentration of 15 mmol / l. The reaction was carried out at RT for 30 minutes. Subsequently, NH ₂ -PEG-Cy 3 ( 42B ) of dye residues by ultrafiltration with MWCO 3000, washed 3 times with 1 ml of 50 mmol / l borate buffer, pH 8, and dissolved in a volume of 200 ul 50 mmol / l borate buffer, pH 8, dissolved.

To this solution was added PDTP-NHS to final concentration of 50 mmol / L. The reaction was carried out at RT for 30 minutes. Subsequently, 50 μl of a 1 mol / l solution of NH ₄ HCO ₃ , pH 8, were added and the reaction mixture was incubated for a further 60 min. To reduce the disulfide bonds, 100 μl of a 1 mol / l TCEP solution, pH 8, was added to the reaction mixture. After 5 min at RT, the product of the reaction, the SH-PEG-Cy3, from the low molecular weight components by ultrafiltration with MWCO 3000, washed 5 times with 1 ml of 50 mmol / l Tris-HCl buffer, pH 7, and dissolved in a volume of 200 μl 50 mmol / l Tris-HCl, pH 7. Immediately before coupling the nucleotide portion, the pH was adjusted to 9.0 with 1 mol / l NaOH.

Coupling of SH-PEG-Cy3 to the nucleotide part:

To 100μl 20 mmol / l dUTP-AA-PDTP in 50 mmol / l borate buffer, pH 9, was added to 100 μl of the solution with SH-PEG-Cy3 in 50 mmol / l Tris-HCl, pH 9.0. Reaction was at RT for 30 min carried out. The product of the reaction was purified by ultrafiltration with MWCO 3000 separated from low molecular weight components, 5 times with 1 ml of 50 mmol / l Tris-HCl buffer, pH 7, and washed in a volume of 200 μl 50 mmol / l Tris-HCl, pH 7, dissolved.

The dUTP-AA-SS-PEG-Cy3 obtained in this way can be isolated from DNA polymerases, for example Klenow fragment exo-minus or Taq polymerase, in the growing strand of nucleic acids are incorporated.

The contains dUTP-AA-SS-PEG-Cy3 a cleavable under mild conditions group, so that the linker can be cleaved with the dye from the nucleotide. This is for example in methods of sequencing by the synthesis of special interest (Balasubramanian WO 03048387, Tcherkassov WO 02088382, Quake WO0132930, Kartalov WO02072892).

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-propargylamino-dCTP, 5-propargylamino-dUTP, 7-deaza-aminopropargyl-dGTP, and 7-deaza-aminopropargyl-dATP may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-deoxyribonuclides can be used, 11 to 14 ,

Example 37:

dUTP-R-CO-S-PEG-Biotin 43

The dUTP-R-COOCH ₃ ( 44 ) was synthesized analogously to the protocol (Heike A. Held, Abhijit Roychowdhury, and Steven A. Benner, Nucleosides, Nucleotides & Nucleic Acids, Vol 22, 391-404 (2003)). The triphosphate synthesis was carried out according to T. Kovacs, L. Ötvös, Tetrahedron Letters, Vol 29, 4525-4588 (1988)).

500 mg (1.41 mmol) of 5-iodo-2'-deoxyuridine were suspended at room temperature in 10 ml of anhydrous DMF under nitrogen atmosphere and 10 min. touched. 160 mg (0.14 mmol) of tetrakis (triphenylphosphine) palladium (0) are then added. After a further 10 minutes, 400 μl of triethylamine, 480 mg (4.28 mmol) of pent-1-methylate and 55 mg (0.29 mmol) of copper (I) iodide are added in succession. After 15 h, the reaction mixture is concentrated on a rotary evaporator and the resulting red oil is purified by chromatography on silica gel (dichloromethane: methanol = 20: 1). 400 mg (84%) of product are obtained. After triphosphorylation, dUTP-R-COOCH _3. Was obtained.

In similar Way you can also cytosine, adenosine and guanosine derivatives with the pent-1-insäuremethylester (Dissertation "Synthesis of base-modified nucleoside triphosphates and their enzymatic polymerization to functionalized DNA ", Oliver Thum, Bonn 2002).

Further modification takes place on dUTP-R-COOCH ₃ :
To 100 .mu.l of a 50 mmol / l solution of dUTP-R-COOCH ₃ in 50 mmol / l borate buffer, pH 9, is added 100 .mu.l of a 1 mol / l solution of mercaptoethanolamine, pH 9, and at 40.degree h stirred. The product of the reaction, the dUTP-R-CO-S-CH ₂ -CH ₂ -NH ₂ ( 45 ) is separated from the excess mercaptoethanolamine on DEAE-cellulose in the borate buffer 10 mmol / l and eluted from the column with 0.3 mol / l NaCl.

This Nucleotide has a thioester group cleavable under mild conditions and may be modified at the amino group on the linker.

At this amino group can further modifications are made.

For example can be coupled to them a dye:

Synthesis of dUTP-R-CO-S-CH ₂ -CH ₂ -NH-R-Cy3 ( 46 )

To 100 μl of a 10 mmol / l solution of dUTP-R-CO-S-CH ₂ -CH ₂ -NH ₂ in 50 mmol / l borate buffer, pH 9, Cy3-NHS was added to a concentration of 15 mmol / l added. The reaction was carried out at RT for 30 min. Subsequently, the Cy3-modified nucleotide on silica gel plate and RP-18 column was purified, similar to that shown in Example 15.

One such nucleotide can be used as a reversible terminator in a method for sequencing nucleic acids can be used (Tcherkassov WO 02088382). The cleavage of the thioester compound For example, by adding 100 mmol / l mercaptoethanol in 50 mmol / l borate buffer, pH 9, take place.

To the amino group of the dUTP-R-CO-S-CH ₂ -CH ₂ -NH ₂ , a long linker can also be coupled with a low molecular weight marker, similar to Example 19. The obtained dUTP-R-CO-S-CH ₂ -CH ₂ -NH-PEG-biotin may serve as an intermediate for a nuc macromolecule.

Example 38:

dUTP-AA-SS-Propionate-TEAE-Cy3-PEG ( 47 )

This Derivative can be prepared from dUTP-AA-SS-propionate-TEAE-Cy3 (see Example 17). by modification of the amino group in the linker with an mPEG-SPA, e.g. 5,000 Da, to be obtained. The modification conditions are similar in Example 19. This molecule has a long linker and can by the method of the invention for rapid purification of the modified nucleotides prior to their use used in the labeling reactions. For a breakup of labeled unlabeled nucleotides may be in this example a filter with MWCO of 3000 can be used.

Example 39:

Production of a modified Klenow fragment Exo minus the DNA polymerase I of E. coli (im Further named as Klenow fragment Exo minus).

In an embodiment of the modification become 70 μl a buffer solution with Klenow fragment exo minus the DNA polymerase (750 units vial from Amersham Bioscience, dissolved in the manufacturer buffer: 50 mmol / l potassium phosphate buffer, pH7, 1.0 mmol / l DTT, 50% glycerol) 100 μl a buffer solution (200 mmol / l Tris-HCl buffer, pH 10.0, 60% glycerol) was added, the pH of the solution with Polymerase is now 9.0. Subsequently be 30 μl a 1 mol / l aqueous Iodine acetamide solution added. The reaction is carried out at RT for 30 min. This is done in this way a selective modification of the polymerase on the SH group of the Cysteine.

In another embodiment the modification takes place first an addition of 10 μl 50 mmol / l TCEP-NaOH solution, pH 8, to 70 μl a buffer solution with Klenow fragment Exo-minus of DNA polymerase (750 units vial from Amersham Bioscience disbanded in the manufacturer buffer, see above). After 10 min at RT then the Add 100 μl a buffer solution (200 mmol / l Tris-HCl buffer, pH 10.0, 60% glycerol) to the solution with Polymerase. Subsequently be 30 μl a 1 mol / l aqueous Iodine acetamide solution added. The Reaction takes place at RT for 30 min. This is a selective Modification of the polymerase on the SH group.

The Purification of the modified polymerase can be carried out, for example, by ultrafiltration or over Ion exchangers or dialysis done.

The Storage of the modified polymerase takes place, for example in a glycerol-containing buffer. Suitable buffers are, for example Tris-HCl, borate, phosphate buffer. The pH of these buffers is for example between 5 and 10. The concentration of glycerol, for example, between 10 and 70% lie.

Other reagents, such as PEG or salts such as NaCl, NH ₄ Cl can be added to the polymerase solution. Preferably, no reducing agent, such as DTT, is added to the polymerase solution. In one embodiment, the storage buffer additionally contains a reagent that can selectively react with SH groups, for example, iodoacetamide in concentration between 1 mmol / l and 500 mmol / l.

A Polymerase modified in this way may be used instead of Klenow fragment Exominus of DNA polymerase used in reactions with nuc macromolecules become.

Example 40:

Synthesis of dUTP-AA-SS-R-PEG-Oligo-dT31-Cy3 ( 48 )

Preparation of SH-R-PEG-Oligo-dT ₃₁ -Cy3 ( 49 ):

To 200 μl of a 100 μmol / l solution of 3'-amino-oligo-dT ₃₁ -Cy3 in 50 mmol / l borate buffer, pH 9, was added NHS-PEG-maleimide until the concentration of 20% (w / v) has been achieved. The mixture was stirred vigorously at 40 ° C for 2 h. The separation of the maleimide-PEG-oligo-dT ₃₁ -Cy3 from the excess of PEG derivative was carried out by DEAE-cellulose column chromatography: The reaction mixture was applied to the column in 10 mmol / l borate, pH 9, and with 20 column volumes of 50 mmol / l borate, pH 9, washed. Elution of maleimide-PEG-oligo-dT ₃₁ -Cy3 from the column was carried out with 1 M NaCl in 50 mmol / l borate, pH 9. The eluate was first concentrated by ultrafiltration (MWCO 3000) and the product was dissolved in 20 mmol / l Borate buffer, pH 9.0, rebuffered. The maleimide-PEG-oligo-dT ₃₁ -Cy3 was separated from oligo-dT ₃₁ -Cy3 on preparative 15% polyacrylic gel by electrophoresis, isolated from the gel and dissolved in 50 mmol / l borate buffer, pH 9.0. Yield: 55%.

To solution with maleimide-PEG-oligo-dT ₃₁ -Cy3, DTT was added to a concentration of 0.5 mol / L and the mixture was stirred at RT for 16 h. Reaction of DTT with maleimide gives rise to SH-R-PEG-oligo-dT ₃₁ -Cy3. This material was separated on DTT excess DEAE column. The reaction mixture was applied to the column in 50 mmol / l Na acetate, pH 6.0, and washed with 20 column volumes of 50 mmol / l Na acetate, pH 6.0. Elution of SH-R-PEG-oligo-dT ₃₁ -Cy 3 from the column was carried out with 1 mol / l NaCl in 50 mmol / l Na acetate, pH 6.0. The eluate was first concentrated by ultrafiltration (MWCO 3000) and the product, SH-R-PEG-oligo-dT ₃₁ -Cy3, was rebuffered in 50 mmol / l borate buffer, pH 9.0. The concentration of SH-R-PEG-oligo-dT ₃₁ -Cy3 was 100 μmol / l.

To this solution were 50 equivalents to dUTP-AA-PDTP (synthesized as in Example 1). To Separation on DEAE-cellulose for 3 h at RT: The mixture was in 50 mmol / l Na acetate buffer, pH 6.0, applied to the column, and with 20 column volumes 50 mmol / l Na-acetate, pH 6.0, washed. Elution of dUTP-AA-PDTP was done with 0.3 mol / l NaCl in 50 mmol / l Na acetate, pH 6.0, elution of dUTP-AA-SS-R-PEG-oligo-dT31-Cy3 carried out with 0.8 mol / l NaCl in 50 mmol / l Na-acetate, pH 6.0. The eluate was with Ultrafiltration (MWCO 3000) first concentrated and the product was dissolved in 50 mmol / l Na acetate, pH 6.0, re-buffered.

The substance carries a nucleoside triphosphate functionality and a macromolecular marker functionalities (oligo-dT31). The oligo-dT31 consists of nucleoside monophosphates, which, however, do not participate in the enzymatic reaction and have only one signal-transmitting function. To such an oligonucleotide complementary nucleic acids can be hybridized, which have a signaling function ( 37B ). (general rules for the hybridization of nucleic acids are known in the art, Anderson "Nucleic Acid Hybridization", 1999.).

The By definition, nucleotide is a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. This connection is derived from polymerases (e.g., Klenow exo minus polymerase and terminal Transferase) as a substrate.

In similar Way you can Also, other oligonucleotides are coupled to the dUTP derivative. In one embodiment of the invention other homopolymer oligonucleotides, e.g. Poly-dC, poly-dG or poly-dU or poly-dA. In another embodiment, too Oligonucleotides with specific sequences can be used. By the specific sequences of oligonucleotides may be nucleic acid chains be hybridized sequence specific. For the synthesis of nuc macromolecules also oligonucleotides are used, the hairpin structures (Stemloops).

In carries this example the oligonucleotide one at the 3 'end via a Left-coupled amino group serving as a coupling site for maleimide-PEG-NHS occurs, and the Cy3 fluorescent dye. Also other coupling groups, such as SH, carboxy, aldehyde groups can be used.

The position of the coupling group may be at one of the ends of the oligonucleotides, or else with lie in the sequence. Such oligonucleotides can be synthesized by the company MWG-Biotech, Germany.

When Modifications can Oligonucleotides fluorescent dyes or other reporter groups, like biotin, carry digaxygenin. Also several modifications per an oligonucleotide are possible. For example, you can FRET pairs or fluorescent dye quanine molecule pair in an oligonucleotide inserted become.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-propargylamino-dCTP, 5-amino-propargyl-dUTP, 7-deaza-aminopropargyl-dGTP, and 7-deaza-aminopropargyl-dATP may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-deoxyribonuclides can be used, 11 to 14 , Other base-modified nucleotides can be used in a similar manner.

By adding poly-dA or poly-A, several dUTP-AA-SS-R-PEG-oligo-dT31-Cy3, eg 10-20, can be coupled to a nuc macromolecule. This results in a nuc macromolecule with linearly arranged nuc-linker components ( 37C ). By adding other modified oligonucleotides which can bind to poly-dA or poly-A, other modifications can also be introduced, eg fluorescence labeling.

Claims (66)

Macromolecular compounds having the structure: (Nuk linker) _n marker wherein: Nuk - is a nucleotide or a nucleoside (Nuk component) Linker - a linker component which includes the following components: a) Coupling unit L - a part of the linker which is the link between Nuk and the linker radical b) a polymer A part of the linker which is a water-soluble polymer with an average length between 100 and 20,000 atoms c) coupling unit T - a part of the linker which represents the link between the linker residue and the marker Marker - a marker component is n - one integer between 1 and 100 is Macromolecular compounds according to claim 1, wherein the nuc-component includes the following structures:

Wherein: Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications, where X is the coupling site of the linker to the base, and L - the link between Nuk and linker R ₁ -is HR ₂ - is independently selected from the group H, OH, halogen, NH ₂ , SH or protected OH group R ₃ - is independently selected from the group H, OH, Hal , PO ₃ , SH, NH ₂ , OR _3-1 , P (O) _m -R _3-1 , NH-R _3-1 , SR _3-1 , Si-R _3-1 wherein R _{3-1 is} a chemical , photochemically or enzymatically cleavable group and m is 1 or 2. R ₄ - is H or OH R ₅ - is independently selected from the group OH, or a protected OH group, or monophosphate group pe, or diphosphate group or triphosphate group Macromolecular compounds according to claim 1, wherein the nuc-component includes the following structures:

Wherein: base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications capable of enzymatic reactions R ₁ - is HR ₂ - independently selected from the group H, OH, Hal, NH ₂ , SH or protected OH group R ₃ - is independently selected from the group OR _3-2 , P (O) _m -R _3-2 , where m is 1 or 2 is NH-R _3-2 , SR _3-2 , Si-R _3-2 or, where R _{3-2 is} the coupling site of the linker to the nucleotide L - the link between Nuk and linker R ₄ - is H or OH R ₅ - is independently selected from the group OH, or a protected OH group, or monophosphate group, or diphosphate group or triphosphate group Macromolecular compounds according to claim 1, wherein the nuc-component includes the following structures:

Wherein: base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications capable of enzymatic reactions R ₁ - is HR ₂ - independently selected from the group H, OH, Hal, NH ₂ , SH or protected OH group R ₃ - is independently selected from the group H, OH, Hal, PO ₃ , SH, NH ₂ , OR _3-1 , P (O ) _m -R _3-1 , NH-R _3-1 , SR _3-1 , Si-R _3-1 wherein R _{3-1 is} a chemically, photochemically or enzymatically cleavable group and m is 1 or 2. R ₄ - is H or OH R ₅ - is independently selected from the group OR _5-1 -L, or P- (O) ₃ -R _5-1 -L (modified monophosphate group), or P- (O) ₃ -P (O) ₃ -R _5-1 -L (modified diphosphate group) or P (O) ₃ -P (O) ₃ P (O) ₃ -R _5-1 -L ( modified triphosphate group), where R _{5-1 is} the coupling site of the linker to the nucleotide and L - is the link between Nuk and linker. Macromolecular compounds according to claims 1 to 4, wherein the coupling unit (L) includes the following structural elements: R ₆ -NH-R ₇ , R ₆ -OR ₇ , R ₆ -SR ₇ , R ₆ -SS-R ₇ , R ₆ - CO-NH-R ₇ , R ₆ -NH-CO-R ₇ , R ₆ -CO-OR ₇ , R ₆ -O-CO-R ₇ , R ₆ -CO-SR ₇ , R ₆ -S-CO- R ₇ , R ₆ -P (O) ₂ -R ₇ , R ₆ -Si-R ₇ , R ₆ - (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -R ₇ , R ₆ - A- (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -BR ₇ , R ₆ - (CH = CH-) _n -R ₇ , R ₆ - (A-CH = CH-) _n - R ₇ , R ₆ - (CH = CH-B-) _n -R ₇ , R ₆ -A-CH = CH- (CH ₂ -) _n -R ₇ , R ₆ - (- CH = CH-CH ₂ ) _n -BR ₇ , R ₆ - (C≡C-) _n -R ₇ , R ₆ - (AC≡C-) _n -R ₇ , R ₆ - (C≡CB-) _n -R ₇ , R ₆ -AC≡C- ( CH ₂ -) _n -R ₇ , R ₆ - (- C≡C-CH ₂ ) _n -BR ₇ , R ₆ - (- C≡C-CH ₂ -CH ₂ -) _n -BR ₇ , wherein R ₆ - the nuc-component is, R ₇ - is the linker radical and A and B include the following structural elements: -NH-, -O-, -S-, -SS-, -CO-NH-, -NH-CO-, -CO-O-, -O-CO-, -CO-S-, -S-CO-, -P (O) ₂ -, -Si-, - (CH ₂ ) _n -, a photolabile group, where n - is equal to 1 to 5 Macromolecular compounds according to claims 1 to 5, wherein the linker component a water-soluble Polymer includes. Macromolecular compounds according to claim 6, wherein the linker component water-soluble Polymers independently selected includes from the following group: Polyethylene glycol (PEG), polysaccharides, dextran, polyamides, polypeptides, polyphosphates, Polyacetates, poly (alkylene glycols), copolymers of ethylene glycol and propylene glycol, poly (olefinic alcohols), poly (vinylpyrrolidones), Poly (hydroxyalkylmethacrylamides), poly (hydroxyalkylmethacrylates), Poly (x-hydroxy acids), polyacrylic acid, polyacrylamide, Poly (vinyl alcohol). Macromolecular compounds according to claims 1 to 7, wherein a linker component an average length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 and 50,000 atoms. Macromolecular compounds according to claims 1 to 8, wherein a marker component a signaling, signal-transmitting, catalytic or affine Function has Macromolecular compounds according to claims 1 to 9, being a marker component consists of a structural marker unit Macromolecular compounds according to claims 1 to 9, being a marker component composed of several structural marker units bound to a core component consists Macromolecular compounds according to claims 10 or 11, wherein a structural marker unit is independent of one includes the following structural elements: Biotin, hapten, radioactive Isotope, rare earth atom, dye, fluorescent dye. Macromolecular compounds according to claims 10 or 11, wherein a structural marker unit is independent of one includes the following elements: Nanocrystals or their modifications, proteins or their modifications, nucleic acid chains or their modifications, particles or their modifications. Macromolecular compounds according to claim 13, wherein a structural marker unit includes one of the following proteins: enzymes or their conjugates or modifications, Antibody or their conjugates or modifications, Streptavidin or his Conjugates or modifications, Avidin or its conjugates or modifications Macromolecular compounds according to claim 13, wherein a structural marker unit one of the following types nucleic acid chains includes: DNA, RNA, PNA, where the length of nucleic acid chains between 10 and 10,000 nucleotides Macromolecular compounds according to claims 11 to 15, wherein the core component of the marker component is independently of the following elements: water-soluble polymer from the group of: polyamides (e.g., polypeptides), polyacrylic acid and its derivatives, polyacrylamides and their derivatives, polyvinyl alcohols and their derivatives, nucleic acids and their derivatives, streptavidin or avidin and their derivatives, dendrimers, these elements being linear or branched or interlinked could be Macromolecular compounds according to claims 1 to 9, 11 to 16, where the connection between several structural Marker units and the core component covalent or affine occurs. Macromolecular compounds according to claims 1 to 10, wherein the compound is between a structural marker unit and the linker is covalent or affine Macromolecular compounds according to claims 1 to 9, 11 to 17, wherein the connection between the core component and the linker is covalent or affine Macromolecular compounds according to claims 1 to 19, with only one nuc-component with a coupled linker component to the marker component is bound, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms. Macromolecular compounds according to claims 1 to 20, wherein only one nuc-component with a coupled linker component to the marker component is bound, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and the linker component one or more compounds which can be cleaved under mild conditions includes. Macromolecular compounds according to claims 1 to 21, with only one nuc-component with a coupled linker component to the marker component is bound, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and one or more parts of the nuc macromolecule so are modified that only a nuk component can be incorporated into a growing strand can. Macromolecular compounds according to claims 1 to 19 wherein several Nuk components each have a linker on a marker components coupled, the length the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms. Macromolecular compounds according to claims 1 to 19, 23, where several nuc-components are each coupled via a linker to a marker component, being the length the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and the respective linker one or more cleavable under mild conditions Includes connections. Macromolecular compounds according to claims 1 to 19, 23, 24, wherein several Nuk components each have a Linker to a marker component coupled, the length the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and a or several parts of the nuc macromolecule modified so are that only a nuc-component can be incorporated into a growing nucleic acid chain. Oligonucleotides or polynucleotides which are at least a nuc macromolecule according to claims 1 to 25 per one nucleic acid chain lock in. Oligonucleotides or polynucleotides according to claim 26, wherein oligo- or polynucleotides are RNA, DNA or PNA whose Length between 5 and 50,000 nucleotides. Method for modifying nucleic acid chains, being for the coupling nuc-macromolecules according to claims 1 to 25 are used The method of claim 28, wherein the modification by an enzymatic coupling and the reaction mixture includes the following components: - at least a type of nuc macromolecule or their intermediates according to claims 1 to 25, wherein each kind the nuc macromolecules one for she has characteristic markings - at least one population the nucleic acid chains, - at least an enzyme type for the coupling of nuc-macromolecules to the nucleic acid chains, The method of claim 28, wherein the modification by an enzymatic coupling and the reaction mixture includes the following components: - at least a type of nuc macromolecule or their intermediates according to claims 1-25, wherein each type of Nuc-macromolecules one for she has characteristic markings - at least one population the nucleic acid chains, - at least a type of enzyme for the coupling of nuc-macromolecules to the nucleic acid chains - at least another type of nucleoside triphosphates The method of claims 29, 30, wherein the enzyme species independently includes one of the following groups: DNA polymerase, RNA polymerase, Terminal transferase, The method of claim 30, wherein the "other type" of the nucleoside triphosphates independently selected is from the group of: ribo-nucleoside triphosphates (ATP, GTP, UTP, CTP), of 2'-deoxyribonucleoside triphosphates (dATP, dUTP, dTTP, dCTP, dGTP), of 2 ', 3'-dideoxynucleoside triphosphates (ddATP, ddGTP, ddUTP, ddCTP, ddTTP). The method of claim 32, wherein the "other type" of nucleoside triphosphates are conventionally modified nucleotides with a label, this label being independent selected is from the group of: a fluorescent dye, a biotin, a Hapten, a radioactive element. Process according to claims 28 to 33, wherein at least two different populations of nucleic acid chains are present The method of claim 34, wherein at least one of the populations of nucleic acid chains has a primer function and at least one pupulation of the nucleic acid chains a matrix function. The method of claim 28, wherein the modification by a chemical coupling, wherein the coupling of nuc-macromolecules to nucleic acid chains by phosphoroamidite coupling takes place. Process according to claims 28 to 36, wherein in the labeling process Nuc-macromolecules are used, which involves coupling only a single nuc-component in the growing nucleic acid strand allow multiple installation by modifications to the Nuk component, and / or the linker component and / or the marker component prevented becomes. The method of claim 37, wherein the further coupling reversibly prevented. The method of claim 37, wherein the further coupling irreversibly prevented. Process according to claims 28 to 36, wherein in the labeling process Nuc-macromolecules are used, the coupling of several nuc-components into the growing nucleic acid strand allow The method of claims 28-40, wherein the at the Reaction participating nucleic acid chains coupled to a fixed phase and addressable positions to have. The method of claim 41, wherein the nucleic acid chains represent a uniform population The method of claim 41, wherein the nucleic acid chains represent two or more different populations and each populations an addressable position on the solid phase Has The method of claims 41.42, wherein the coupling of nuc macromolecules at one Population of uniform nucleic acid molecules fixed to the solid phase, wherein the marker component of the nuc macromolecule is elongated after coupling nucleic acid strand remains and is not separated. The method of claims 41.42, wherein the coupling of nuc macromolecules at one Population of uniform nucleic acid molecules fixed to the solid phase, wherein the marker component or its individual components with or without linker component of the nuc macromolecule during coupling or after the coupling is separated from the incorporated in the growing nucleic acid strand Nuk component becomes. The method of claims 41.43, wherein the coupling of nuc macromolecules in one Reaction batch parallel to two or more different Populations on the solid phase of fixed nucleic acid molecules take place, these populations each being differently addressable Have positions on the solid phase and the marker component of the Nuc-macromolecule after coupling to the extended nucleic acid strand remains and is not separated. A method according to claims 41.43, wherein the coupling of nuc-macromolecules in a reaction batch parallel to two or more different populations of the fixed phase of fixed nucleic acid molecules, these populations have different addressable positions on the solid phase and the marker component or its individual components is separated with or without linker component of the nuc macromolecule during coupling or after coupling of the nuc-component. The method of claims 41 to 47, wherein the addressable Positions with nucleic acid molecules on the solid phase distributed as spots on a flat surface are, where per one spot nucleic acid molecules are uniform. The method of claims 41 to 47, wherein the addressable Positions with nucleic acid molecules to the globule or particles are attached, wherein for a bead nucleic acid molecules are uniform. The method of claims 41 to 47, wherein the addressable Positions with nucleic acid molecules in one Multi vascular system, like microtiter plate or nanotiter plate or picotiter plate, are distributed, wherein in a vessel of the multi-vascular system the nucleic acid molecules uniform are. Process according to claims 28 to 35 and 37 to 50, which includes the following steps: a) Providing at least one population of single-stranded nucleic acid chains b) Hybridization of primers to these nucleic acid chains, with extension-capable NSK primer complexes c) Incubation of at least one type of nuc macromolecule after claims 1 to 25 together with a kind of the polymerase according to claim 31 with the NSK primer complexes provided in steps a and b under conditions the incorporation of complementary Allow nuc-macromolecules, wherein each type of nuc macromolecule has a characteristic mark for them has. d) Removal of unincorporated nuc macromolecules from the NAC primer complexes e) Detection of signals from incorporated into the NSK primer complexes Nuc-macromolecules f) Removal of Linker Component and Marker Component from the nuc-macromolecules incorporated into the NSK primer complexes G) Wash the NSK primer complexes if necessary repetition of steps (c) to (g). The method of claims 28-40, wherein the nucleic acid chains to a solid phase in random Arrangement are coupled. The method according to claims 28 to 41, 52 for the parallel sequence analysis of nucleic acid sequences (nucleic acid chains, NSKs), wherein fragments (NSKFs) of single-stranded NSKs with a length of about 50 to 1000 nucleotides, which can represent overlapping partial sequences of a total sequence, one the NSKFs using a single or multiple different primers in the form of NSKF primer complexes on a reaction surface in a random array, one carries out a cyclic assembly reaction of the complementary strand of the NSKFs using one or more polymerases by reacting a) to those described on the Surface-bound NSKF-primer complexes, a solution containing one or more polymerases and one to four nuc-macromolecules having a labeled with fluorescent dyes marker component, wherein the simultaneous use of at least two nuc-macromolecules to each of the markers Component bef inductive fluorescent dyes are selected so that the nuc-macromolecules used can be distinguished from each other by measuring different fluorescence signals, wherein the nuc-macromolecules are structurally modified so that the polymerase after incorporation of such a nuc-macromolecule in a growing complementary strand is not able is to incorporate another Nuk nacromolecule in the same strand, wherein the linker component with the marker component are cleavable, b) incubating the obtained in step a) stationary phase under conditions suitable for extending the complementary strands, wherein in each case, the complementary strands are extended by one nuc-macromolecule; c) the stationary phase obtained in stage b) is washed under conditions which are suitable for removing nuc-macromolecules not incorporated into a complementary strand, d) the individual, in complementary Strands of incorporated nuc-macromolecule e detected by measuring the signal characteristic of each fluorescent dye, while simultaneously determining the relative position of the individual fluorescence signals on the reaction surface, one e) to generate unlabelled (NTs or) NSKFs, cleave the linker moiety and the label moiety from the nucleated components attached to the complementary strand, f) washing the stationary phase obtained in step e) under conditions sufficient to remove the labels Component are suitable, the steps a) to f) optionally repeated several times, wherein the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs by specific assignment of the in step d) in successive cycles to the respective Positions detected fluorescence signals to the nuc-macromolecules determined. A method according to claim 53, characterized that the steps a) to f) of the cyclic synthesis reaction several times repeated, wherein in each cycle only one type of nuc-macromolecules used. A method according to claim 53, characterized that the steps a) to f) of the cyclic synthesis reaction several times repeated, with two different ones in each cycle labeled types of nuc macromolecules. A method according to claim 53, characterized that the steps a) to f) of the cyclic synthesis reaction several times repeated, each with four different in each cycle labeled types of nuc macromolecules. A method according to claim 53, characterized that the NSKs are variants of a known reference sequence and one the steps a) to f) of the cyclic synthesis reaction several times repeated, where in the cycles alternately two different labeled types of nuc macromolecules and two unlabelled NTs and one uses the total sequences by comparison with the reference sequence determined. Process according to Claims 53 to 57, characterized that in each case a Primerbindungsstelle (PBS) is introduced into the NSKFs, where one with double-stranded one NSKs on both complementary single strands respectively introduce a PBS and wherein the primer binding sites are the same or the same for all NSKFs have different sequences. Process according to Claims 53 to 57, characterized that one contacts the NSKFs with primers in a solution under conditions which leads to the hybridization of the primer to the primer binding sites (PBSs) of the NSKFs are suitable, the primers being identical to one another or different sequences, and the NSKF primer complexes formed subsequently binds on the reaction surface. Process according to Claims 53 to 57, characterized that you first open the NSKFs the reaction surface immobilized and only afterwards with primers under conditions that bring about hybridization the primer to the primer binding sites (PBSs) of the NSKFs suitable wherein NSKF primer complexes are formed, wherein the primers have mutually identical or different sequences. Process according to claims 53 to 60, wherein 10 to 100,000 different sequences populations the incorporation reaction carried out in parallel Process according to claims 53 to 60, wherein 100,000 to 100,000,000 different sequence populations the, installation reaction carried out in parallel. The method of claims 28 to 62, wherein the sequences the nucleic acid chains be determined The method of claims 28 to 63, wherein the marker component fluorescently labeled Process according to claims 41 to 64, wherein the solid Phase independent selected from the following group is: silicone, glass, ceramics, plastics, gels or their modifications A kit that mimics the macromolecular compounds claims 1 to 25.