DE10046184A1 - Universally applicable method for parallel detection of nucleic acids, by forming a primer extension product that includes single-stranded segment for specific hybridization - Google Patents

Abstract

Parallel detection of nucleic acids (16) by a reaction that generates a nucleic acid with a single-stranded (ss) segment. Compounds (14) are prepared, each comprising first primer (10), specific for (16), and an identification sequence (12), specific for (10). (12) is one of a group of sequences that, under predetermined, uniform hybridization conditions, do not cross-hybridize with either each other or (10), and (14) includes an agent that ensures formation of an ss segment during subsequent reaction. (16) is treated with (14) and with second primer (18), specific for (16), that is one of a group of sequences that, under the hybridization conditions, do not cross-hybridize with (12), and polymerase extension of (14) and (18) is performed to produce a hybrid comprising a double-stranded (ds) segment and an ss-segment (XX) that is (12) or its complement. The products are separated from non-extended compounds and treated with a binding sequence (26), immobilized on carrier (22), under conditions such that the ds region of the product is retained. (26) is selected from a group of sequences that, under the hybridization conditions, do not cross-hybridize with an (XX) that is not exactly complementary. Hybridization to (26) is detected. An independent claim is also included for a kit for the new method.

Description

The invention relates to a method for detection at least a nucleic acid sequence, a use and a kit for Implementation of the method according to the invention.

For the detection of a nucleic acid sequence, it is generally be knows the nucleic acid sequence to be detected by means of spec Fischer DNA primer in a polymerase chain reaction (PCR) to reproduce. The duplicated sequences can then by hybridization with one for the after pointing nucleic acid sequence specific binding sequence be detected. With this method, it is disadvantageous that a specific for each nucleic acid sequence to be detected binding sequence is required. Furthermore, it is from Disadvantage that for each hybridization with this binding specific hybridization conditions are observed have to.

From Whitcombe, D. et al., Nature Biotechnology 17 (1999) Sci It is known from 804 to 807 to use a special PCR Perform primer. The primer has an area in which is the synthesis of a counter strand by a blocking agent for a polymerase is prevented. The area instructs Fluorophore and a quencher, which is formed by bases pairings are in the immediate vicinity. A fluo Resence signal cannot be generated. The area points in a non-base paired section a sequence that complementary to one by extending the primer is the product of the PCR. After denaturation of the product, a condition occurs under suitable conditions Base pairing of the section with the complementary area in the product. This causes the fluorophore to quencher  separates so that a fluorescence signal can arise. The Fluorescence signal serves as proof of the presence a DNA sequence specific for the primer. When ver driving it is disadvantageous that a Nu to be verified for each small acid sequence specific primer with a specific complementary section must be synthesized. Through the the primer behaves different functional units long and therefore expensive to manufacture.

It is known from EP 0 401 037 for a PCR nucleotide to be used in the nucleic acid synthesis to be reproduced quenz does not occur. Such a nucleotide can be deoxy Be uridine. After a replication reaction, a unwanted product treated with uracil DNA glycosylase become. Uracil DNA glycolyase cleaves the gycosidic bin between the base uracil and the sugar deoxy-ribose a deoxy-uridine residue built into a DNA molecule. This can cause the unwanted product in another ver diversification reaction no longer serve as a template. On Another nucleotide that can be used is bromodeoxy-uridine. bromine DNA containing deoxy-uridine can be treated with light be dismantled under suitable conditions.

From US 5,744,311 a on a strand displacement ba reproducing method for nucleic acids known. Here, a primer is ver with the 3 'end diverse single-stranded nucleic acid hybridized and extended by means of a DNA polymerase. To extend Deoxy nucleoside triphosphates are used, some of which are derivatized. Suitable derivatized deoxy nucleos sidetriphosphates are e.g. B. α-thio-deoxy nucleoside triphospha te. A strand of the resulting double-stranded DNA with de rivatized deoxy nucleotide residues are used on certain  Sites recognized by a restriction endonuclease and ge cut. Starting from the cut, the DNA extends Polymerase the 3 'end of the cut DNA strand. The one the part of the cut DNA strand is double ousted stranded DNA. By repeating the procedure the displaced DNA strand is duplicated.

From WO 97/31256 it is known to target sequences in nuclein acids by means of a ligase reaction and an immobilized Addressable matrix having binding sequences point. There is one for each target sequence to be verified Oligonucleotide probe used that has a target sequence specific and a binding sequence specific portion having. Furthermore, a marking substance is shown send second oligonucleotide probe used on the target sequence to be demonstrated in the immediate vicinity of the first oligonucleotide probe. If available its the target sequence bind the first and the second oligonu kleotide probe at the target sequence. You are going through a league se covalently linked. The connected oligonu Kleotide probes are used with the immobilized binding sequences zen brought into contact so that hybridization takes place det. The presence of the target sequence is determined by the detec animals of the labeling substance at the site of hybridization demonstrated. The disadvantage of the method is that it often gives false positive results.

The object of the present invention is to overcome the disadvantages to eliminate the state of the art. In particular, a Process, use and a kit for proof at least at least one nucleic acid sequence can be given.  

The object is achieved by the features of claims 1, 2, 42, 43, 44, 45, 47, 48, 49, 52, 53 and 56 solved. expedient Embodiments of the invention result from the features of claims 3-41, 46, 50, 51, 54, 55 and 57-63.

According to the invention, a method for the detection of at least one nucleic acid sequence by means of a reaction producing a counter strand to the nucleic acid sequence is provided with the following steps:

• a) Providing a connection formed from a for the nucleic acid sequence specific to be detected first Primer and an Identifi specific for the first primer Ornamental sequence, with an agent being present in the compound hen, which is a synthesis of a Ge in the reaction prevented against the identification sequence,
• b) contacting the connection with the one to be verified Nucleic acid sequence and a second for the to be detected Nucleic acid sequence specific primer,
• c) performing the reaction, using both the second primer as well as the connection by means of a polymerase reaction mo be differentiated so that a specific product is formed is composed of a double-stranded area and the single-stranded identification sequence,
• d) contacting the modified compound with a to at least partially complete the identification sequence binding sequences immobilized on a carrier, wherein the identification sequence specific with the binding site hybridized and  
• e) detection of hybridization of the modified compound with the binding sequence.

Furthermore, a method for the detection of at least one nucleic acid sequence by means of a reaction producing a counter strand to the nucleic acid sequence is provided with the following steps:

• a) Providing a connection formed from a for the nucleic acid sequence specific to be detected first Primer and an Identifi specific for the first primer zierungssequenz,
• b) contacting the connection with the one to be verified Nucleic acid sequence and a second for the to be detected Nucleic acid sequence specific primer,
• c) performing the reaction, using both the second primer as well as the connection by means of a polymerase reaction mo be differentiated, and a degradation reaction, so that a speci fish product is formed consisting of a double stringy area and at least part of the Identification sequence or at least from part of de existing single strand area,
• d) contacting the single-stranded area with a to at least partially complementary to a carrier immo bilized binding sequence, the single-stranded Be hybridizes richly with the binding sequence and
• e) Evidence of hybridization of the single-stranded Be rich with the binding sequence.

Under a primer, one is extended by a polymerase understandable oligonucleotide. The primers can be made from DNA consist. Speci for the nucleic acid sequence to be detected The first and second primers are primers which are suitable under specific hybridization conditions with the after pointing nucleic acid sequence or a complementary one Hybridize the opposite strand. A speci for the first primer fish identification sequence is an identification sequence sequence that is clearly assigned to the first DNA primer. The agent can be used within the identification sequence, internally half of the first primer or between the first primer and the identification sequence.

A specific product is a product which is specific to the nucleic acid sequence to be detected is. So that such a product is formed at Step lit. c stringent conditions are chosen.

Under a degradation reaction, one becomes the identifier quenz or its counter-strand-modifying reaction understood the. It separates the identification sequence or the opposite strand of the respectively unmodified complex mental strand. The degradation reaction can only be a single one Bindings in the identification sequence or the counter strand divisive reaction. Such a reaction can lead to a Fragmentation of the identification sequence or the Ge lead opposite strand. The hybrid from the identification sequence and the opposite strand is thus destabilized thermodynamically lized that it is the latest among those for step lit. d required such stringent conditions dissociate.

At step lit. d is the specific hybridization of the Identification sequence or the single-stranded area  with the binding sequence under suitable stringent conditions conditions. These conditions depend on the sequences of the Identification sequence or the single-stranded area and the binding sequence. Identifying such conditions is the expert, for. B. from the determination of the hybridisie conditions for PCR primers known.

The detection of hybridization can e.g. B. by proof one with the single-stranded area or the modified Connection related labeling substance.

The advantage of the method is that the formed specific product directly with the immobilized binding sequence can hybridize without being denatured beforehand have to. It is also advantageous in the method that is used for the reaction of a polymerase. A sol The reaction is more specific than a ligase reaction, Wei it is also advantageous that a carrier with immobilized Binding sequences multiple times and for the detection of different nu Small acid sequences can be used. Predefined iden Identification sequences can be in different Verification method with different first primers ver be bound. Identical binding sequences can then be used for the Detection of different nucleic acid sequences used become.

It is advantageous in a process with a degradation reaction liable if the identification sequence or its opposite strand has at least one nucleotide sequence which is an enzy Matic degradation reaction allowed. It is particularly advantageous if the identification sequence is at least one nucleotide contains or in the opposite strand at least one nucleotide is installed, which enables a degradation reaction. The  Nucleotide can be a deoxy uridine residue, a bromodeoxy uridine residue Residue or an α-thio-deoxy nucleotide residue. The mines action can be done with uracil DNA glycosylase, under light effect or by means of an identification sequence or the restriction strand-cloning restriction endonuclease, in particular especially BsrI, BstNI, BsmAI, BsII, BsoBI or BstOI leads.

The reaction can be a polymerase chain reaction (PCR). The first primer is preferably at its 5'-terminal Be rich, especially its 5 'end, with identification sequence connected. This facilitates modification by the Polymerase reaction. To provide the connection the first primer and the identification sequence using ei ner, in particular the agent-forming coupling group ver be bound. To do this, the first primer or the Identifi ornamentation sequence have an activated coupling group. The agent can lead to termination of the polymerase reaction be a means. The agent preferably consists of min at least one nucleotide analog, RNA, PNA or a sub refraction of the sequence of the nucleotides in the compound connecting phosphodiester bonds, in particular by ei a polyethylene glycol linker, a peptide bond or a Disulfide bond. In one embodiment, step lit. c in contact with the immobilized binding sequence carried out. This has the advantage that the individual strands area immediately in contact with the Binding sequence is located and step lit. d therefore no to additional work step required. Hybridizing the single stranded region with the binding sequence can be selected during the execution of step lit. c are tracked. This also makes it possible to quantify those to be verified Nucleic acid sequence.  

At step lit. d can be the product or a fragment of the Product consisting of at least part of the counter strand the identification sequence or from at least part of the double-stranded area and at least part of the Ides tification sequence in contact with the binding sequence be brought. The identification sequence and / or the bin can be a nucleic acid, a nucleic acid derivative or an analog of a nucleic acid or a nucleic acid derivatives, in particular PNA, RNA or modified DNA, ent hold. The identification sequence preferably has 5 to 20 bases on.

The carrier can be made of glass or plastic. before it is in the form of an identifiable particle, egg ner membrane or a chip. The binding sequence can directed and / or immobilized on the carrier via a linker be based. The binding sequence to egg is advantageously immobilized ner defined point of the carrier.

Identify those hybridized with the binding sequence modified compound or with the binding sequence hy bridized single-stranded area can by a Lokali the hybridization on the carrier or an identifi cation adorn the wearer. This allows identification can be carried out very quickly and easily. The Trä lies ger z. B. in the form of a membrane and is the modified Marked compound using a dye, so it can Identify the mo hybridized with the binding sequence identified compound by localizing the dye done on the membrane. The carrier can ver up to 1000 have different binding sequences.  

In a preferred embodiment, the second primer or one by means of the polymerase reaction on the first or nucleotide attached to the second primer is a labeling sub punch on. The marking substance can be optically detectable animal substance, in particular a fluorophore or a color substance, a, especially radioactive, isotope, a hapten, a particle, a sub detectable by plasmon resonance punch, a ligand, especially biotin, or a reducible re or oxidizable substance. It is preferably the fluorophore is FAM (5 [6] -carboxyfluorescein), JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), ROX (5 [6] -carboxy-X-rhodamine) or TAMRA (carboxytetramethylrho Damin). The product can be customized with a double strand Intercalator, especially that from Molecular Pro bes, Inc. Eugene, OR 97402-0469, USA distributed SYBR Green, be marked. In step lit. e the marking substance or the one stored in the product Intercalator detected.

The nucleic acid sequence to be detected can be a DNA. It can also be an RNA, whereby the ver binding in step lit. b additionally with a reverse Transcriptase is contacted and in step lit. c a reverse transcription is also carried out. In step lit. b an additional third for the Detected nucleic acid sequence of specific primers are set, at which in step lit. c by means of the Re verse transcriptase nucleotides can be added. Preferably the nucleic acid sequence to be detected is double-stranded before and before or during step lit. b melted. The after completing step lit. c if necessary in addition to the modifi graced connection still existing unmodified connection dung can be taken before step lit. d, especially using sili  Ca particles, glass particles or a suitable filter removed become. To remove the unmodified connection the individual in the prior art for unspecific removal known stranded oligonucleotides. The Removing the unmodified connection prevents a Occupying binding sequences through this connection. It can increase the sensitivity of the procedure. Preferably is the amount of binding se immobilized on the support quenz at least as large as the amount of total ready made connection. The after step lit. c before existing unmodified connection then need not ent to be removed to increase the sensitivity of the method hen.

The first and second primers are preferably selected so that that a from the first primer and the Nu hybrid formed essentially the same Melting temperature is like that of the second primer and the opposite strand of the nucleic acid sequence to be detected formed hybrid. In a preferred embodiment are the first and second primers, the identifiers quenz and the binding sequence chosen so that the enamel point of a hybrid from the nucleic acid to be detected quenz and the first primer or a hydride from the Ge gene strand of the nucleic acid sequence to be detected and the second primer at least 5 ° C, especially 10 ° C, above the melting point of a hybrid from the identification sequence and the binding sequence or from the opposite strand of Identification sequence and the binding sequence. The is particularly advantageous if step lit. c in Contact with the immobilized binding sequence performed becomes. Then by means of an appropriate temperature control step lit. c be carried out without a hy  bridization with the binding sequence adversely affects become. The hybridization can then be carried out after the Step lit. c can be achieved by cooling.

If multiple nucleic acid sequences are to be detected len, the binding sequences can be chosen so that ver separated from the identification sequences and the binding sequences formed specific hybrids essentially identi have melting temperatures. This has the advantage that all hybridizations under uniform conditions can be performed and thus the proof of many nucleotides acid sequences is facilitated in one operation. The Identification sequences, the binding sequences and the he Most, second and possibly third primers are preferably so chosen to be among their respective stringent hybridi conditions do not form unspecific hybrids. If several nucleic acid sequences are to be detected it is advantageous if the first and second primers are chosen in this way are that different from the first primers and the after pointing nucleic acid sequences and the second primers and the counterstrands of the nucleic acid sequences to be detected specific hybrids formed an essentially identical one Have melting temperature. The reaction can then for meh higher nucleic acid sequences under identical conditions be performed. Preferably the first and second Primer selected so that in step lit. c products formed which are essentially the same length. The products are then formed in similar amounts and behave thermodynamically almost identical. This contributes to the unity process conditions.

In a method for the detection of multiple nucleic acid sequences The first primer and / or the identification can  tion sequence for several of the nucleic acid to be detected sequences are specific. Is one of the second primers thereby specifying for one of the nucleic acid sequences to be detected zifisch and has a specific for this second primer Marking substance. At step lit. e is done additionally Lich identifying the specific labeling substance Zen. The labeling substances must be clearly different from each other be distinguishable.

In a method for the detection of multiple nucleic acid sequences steps z. a, b and c with different Detect nucleic acid sequences separately using distinguishable labeling substances and / or intercalators and the steps lit. d and e common be carried out sam. At step lit. e then takes place additionally identifying the labeling substances and / or intercalators.

According to the invention, an identification is also used Ornamental sequence provided, the identification sequence at least one a synthesis of a counter strand of the identifi ornamental sequence preventing agent or wherein the Identification sequence nucleotides and / or a nucleotide quenz contains, which allows / allow a degradation reaction. Another use is an activated coupling group having first primers or an activated one Coupling group having identification sequence vorese hen. Furthermore, the invention relates to the use of a from a first primer and one for the first primer specific identification sequence formed connection where a means is provided in the connection, which means the reaction a synthesis of a counter strand to the identifi Prevents or the identification sequence  contains nucleotides and / or a nucleotide sequence, wel allow / allow a degradation reaction.

Furthermore, the use of a second primer in one inventive method provided. The second can Primers have a labeling substance. Furthermore concerns the invention the use of with a marking sub punched nucleotides in the reaction. Erfindungsge The use of a double strand is also appropriate Intercalators, especially SYBR Green, for marking the Product. The invention further relates to the use of a having at least one immobilized binding sequence Carrier. The binding sequence can be at a defined point of the carrier be immobilized. The carrier can also be a be an identifiable carrier. The carrier can be made of glass or Plastic and / or in the form of an identifiable Particles, a membrane or a chip are present. OF INVENTION according to the use of a polymerase and / or a reverse transcriptase. It is understood that also an analogous to the procedural execution play the invention designed uses fiction according to.

A kit for carrying out a method according to the invention is also provided, comprising:

• a) a binding sequence immobilized on a support and
• b) one at least partially complete with the binding sequence mental identification sequence, the identification sequence has a means which ei after coupling  the first primer and the identification sequence in the Reaction a synthesis of a counter strand for identification sequence prevented or
• c) an identification sequence which itself or its counter strand that can be synthesized in the reaction, at least is partially complementary to the binding sequence, the Identification sequence nucleotides and / or a nucleotide quenz contains, which allows / allow a degradation reaction.

The identification sequence can be an activated coupling Group for coupling the 5'-terminal region, in particular of the 5 'end, of the first primer. In the kit can a first primer may be included, either the Identi fication sequence or the first primer an activated Coupling group for coupling the 5'-terminal region, ins particular of the 5 'end, the first primer.

A kit is also provided, containing:

• a) a binding sequence immobilized on a support and
• b) a connection formed from one for the evidence de nucleic acid sequence specific first primer and one for the first primer specific to the binding sequence at least partially complementary identification sequence, where a means is provided in the connection, which means the reaction a synthesis of a counter strand to the identifi ornamental sequence prevented  or
• c) a connection formed from one for the evidence de nucleic acid sequence specific first primer and one for the first primer specific identification sequence, the identification sequence being nucleotides and / or a Contains nucleotide sequence, which he a degradation reaction leaves / allow and where the identification sequence or de ren at least synthesizable in the reaction is partially complementary to the binding sequence.

The binding sequence is preferably at a defined position of the carrier immobilized. The carrier can also be an identifi decorative carrier. The carrier advantageously consists made of glass or plastic and / or lies in the form of an ident detectable particle, a membrane or a chip. The agent can be composed of at least one nucleotide analog, RNA, PNA or an interruption in the sequence of the nucleotides in the compound connecting phosphodiester bonds, ins especially through a polyethylene glycol liner, a peptide bond or a disulfide bond. In the kit can contain a polymerase and possibly a reverse transcriptase his. In addition, a, in particular a marker substance-containing, second primer may be included. The kit can also be labeled with nucleotides contain. Furthermore, a double strand specific interka lator, in particular SYBR Green, may be included. It understands itself that an analogous to the procedural Ausfüh Example of the invention designed kit fiction according to.  

Exemplary embodiments of the invention are described below described the drawing. Show here:

Fig. 1a, b show a schematic representation of the Vervielfälti supply a nucleic acid sequence to be detected by means of a PCR,

Fig. 2a-c is a schematic representation of ger on a Trä immobilized binding sequences and their hybridization with a compound

Fig. 3 is a schematic representation of the amplification of an RNA RNA by means of a reverse transcript and a DNA polymerase in a flow chart and

Fig. 4 is a schematic representation of a duplication of a nucleic acid sequence to be detected by means of a PCR and a subsequent degradation reaction.

A connection 14 composed of a first primer 10 and an identification sequence 12 is shown in FIG. 1a. The primer 10 binds to the nucleic acid sequence 16 to be detected. The second primer 18 has a marking substance 20 . It binds to the opposite strand of the nucleic acid sequence 16 to be assigned. In Fig. 1b the result of a PCR is shown schematically, which has been carried out with the compound 14 shown in Fig. 1a, the second primer 18 and the nucleic acid sequence 16 to be detected. The identification sequence 12 contains means which prevent the region of the identification sequence 12 from becoming double-stranded. Products with a double-stranded area and the single-stranded identification sequence were created.

Fig. 2a shows a support 22. It can be z. B. act on egg ne membrane. On the carrier 22 24 binding sequences 26 are bound Linker. In Fig. 2b shows the situation after the contacting is illustrated in Fig. 2a are complementary to the identifying sequences 12 binding sequences 26 is shown with the illustrated in Fig. 1b PCR products. The identification sequences 12 bind to the binding sequences 26 . The hybridization on the carrier 22 can be detected by the marking substance 20 . Fig. 2c does not show the situation after contacting the presented in Fig. 2a Darge binding sequences 26 with Polymer A by a se-modified reaction connections 14. Such a situation arises if the nucleic acid sequence 16 to be detected is not present. The identification sequences 12 bind to the binding sequences 26 . A labeling substance 20 cannot be detected at the site of the hybridization because no PCR product containing the second primer 18 with the labeling substance has been produced.

Fig. 3 schematically shows a method in which the nachzu facing nucleic acid sequence 16 is a RNA. First, the second primer 18 containing the labeling substance 20 binds to the RNA. It is extended using a reverse transcriptase. This creates a double-stranded DNA-RNA hybrid. After the DNA-RNA hybrid has been denatured, the first primer 10 binds to the DNA strand. A PCR is carried out to amplify the DNA strand.

Fig. 4 shows a composed of a primer 10 and a sequence iden approximately 12 Compound 14. The identification sequence 12 has nucleotides 13 which can be modified by a degradation reaction. A PCR reaction with this compound, a nucleic acid sequence 16 to be detected and a second primer 18 leads to completely double-stranded PCR products. The identification sequences contained therein are removed by a degradation reaction. Double-stranded products with a single-stranded counter strand of the identification sequence result.

LIST OF REFERENCE NUMBERS

10

first primer

12

identification sequence

13

modifiable nucleotide

14

connection

16

nucleic acid sequence

18

second primer

20

marker

22

carrier

24

left

26

binding sequence

Claims (63)

1. A method for the detection of at least one nucleic acid sequence ( 16 ) by means of a reaction producing a counter strand to the nucleic acid sequence ( 16 ) with the following steps:

• a) providing a compound ( 14 ) formed from a first primer ( 10 ) specific for the nucleic acid sequence ( 16 ) to be detected and a specific identification sequence ( 12 ) for the first primer ( 10 ), an agent being present in the compound ( 14 ) is provided which prevents synthesis of a counter strand to the identification sequence ( 12 ) during the reaction,
• b) bringing the compound ( 14 ) into contact with the nucleic acid sequence ( 16 ) to be detected and a second primer ( 18 ) specific for the nucleic acid sequence ( 16 ) to be detected,
• c) carrying out the reaction, both the second primer ( 18 ) and the compound ( 14 ) being modified by means of a polymerase reaction, so that a specific product is formed, consisting of a double-stranded region and the single-stranded identification sequence ( 12 ),
• d) contacting the modified compound (14) with a to the identifying sequence (12) at least partially complementary to a support (22) immobilized binding sequence (26), the identifying sequence (12) hybridized to specific fish with the binding sequence (26) and
• e) detection of hybridization of the modified compound ( 14 ) with the binding sequence ( 26 ).

2. Method for the detection of at least one nucleic acid sequence ( 16 ) by means of a reaction producing a counter strand to the nucleic acid sequence ( 16 ) with the following steps:

• a) providing a compound ( 14 ) formed from a first primer ( 10 ) specific for the nucleic acid sequence ( 16 ) to be detected and an identification sequence ( 12 ) specific to the first primer ( 10 ),
• b) bringing the compound ( 14 ) into contact with the nucleic acid sequence ( 16 ) to be detected and a second primer ( 18 ) specific for the nucleic acid sequence ( 16 ) to be detected
• c) performing the reaction, wherein both the second primer ( 18 ) and the compound ( 14 ) are modified by means of a polymerase reaction, and a degradation reaction, so that a specific product is formed, consisting of a double-stranded region and at least one of part of the identification sequence ( 12 ) or at least part of its counter-strand consisting of single-stranded regions,
• d) bringing the single-stranded region into contact with an at least partially complementary binding sequence ( 26 ) immobilized on a support ( 22 ), the single-stranded region specifically hybridizing with the binding sequence ( 26 ) and
• e) Detection of the hybridization of the single-stranded region with the binding sequence ( 26 ).

3. The method according to claim 2, wherein the identification sequence ( 12 ) or the opposite strand has at least one nucleotide sequence which allows an enzymatic degradation reaction. 4. The method according to claim 2 or 3, wherein the identification sequence ( 12 ) contains at least one nucleotide ( 13 ) or in the opposite strand at least one nucleotide ( 13 ) is incorporated, which enables a degradation reaction. 5. The method according to claim 4, wherein the nucleotide ( 13 ) is a deoxy-uridine residue, a bromodeoxy-uridine residue or an α-thio-deoxy nucleotide residue. 6. The method according to any one of claims 2-5, wherein the degradation reaction by means of uracil DNA glycosylase, under the action of light or by means of a restriction endonuclease which cleaves the identification sequence ( 12 ) or its counter-strand, in particular BsrI, BstNI, BsmAI, BsII, BsoBI or BstOI, is carried out. 7. The method according to any one of the preceding claims, wherein the reaction is a polymerase chain reaction (PCR). 8. The method according to any one of the preceding claims, wherein the first primer ( 10 ) at its 5'-terminal region, in particular its 5'-end, is connected to the identification sequence ( 12 ). 9. The method according to any one of the preceding claims, wherein for providing the connection ( 14 ), the first primer ( 10 ) and the identification sequence ( 12 ) are connected by means of a coupling group, in particular forming the agent. 10. The method according to claim 9, wherein the first primer ( 10 ) or the identification sequence ( 12 ) has an activated coupling group. 11. The method according to any one of the preceding claims, wherein the agent leads to termination of the polymerase reaction Is medium. 12. The method according to any one of the preceding claims, wherein the agent at least from a nucleotide analog, RNA, PNA or an interruption of the sequence of the phosphodiester bonds connecting the nucleotides in the compound ( 14 ), in particular by a polyethylene glycol linker, a peptide bond or a disulfide bond , consists. 13. The method according to any one of the preceding claims, wherein step lit. c is carried out in contact with the immobilized binding sequence ( 26 ). 14. The method according to any one of the preceding claims, wherein in step lit. d the product or a fragment of the product, consisting of at least part of the counter-strand of the identification sequence ( 12 ) or of at least part of the double-stranded region and at least part of the identification sequence ( 12 ), is brought into contact with the binding sequence ( 26 ) , 15. The method according to any one of the preceding claims, wherein the identification sequence ( 12 ) and / or the binding sequence ( 26 ) contains a nucleic acid, a nucleic acid derivative or an ana logon of a nucleic acid or a nucleic acid derivative, in particular PNA, RNA or modified DNA. 16. The method according to any one of the preceding claims, wherein the identification sequence ( 12 ) has 5 to 20 bases. 17. The method according to any one of the preceding claims, wherein the carrier ( 22 ) consists of glass or plastic. 18. The method according to any one of the preceding claims, where in the carrier ( 22 ) is in the form of an identifiable Parti kels, a membrane or a chip. 19. The method according to any one of the preceding claims, where the binding sequence ( 26 ) is directed and / or immobilized on the carrier ( 22 ) via a linker ( 24 ). 20. The method according to any one of the preceding claims, wherein the binding sequence ( 26 ) is immobilized at a defined location of the carrier ( 22 ). 21. The method according to any one of the preceding claims, wherein identifying the the binding sequence (26) hybridized overbased modified compound (14) or of the extension sequence with the bin (26) hybridized single-stranded region by locating the hybridization on the support (22) or the carrier ( 22 ) is identified. 22. The method according to any one of the preceding claims, wherein the carrier ( 22 ) has up to 1000 different binding sequences ( 26 ). 23. The method according to any one of the preceding claims, wherein the second primer ( 18 ) or a nucleotide added to the first ( 10 ) or the second primer ( 18 ) by means of the polymerase reaction has a labeling substance ( 20 ). 24. The method according to claim 23, wherein the marking substance ( 20 ) is an optically detectable substance, in particular a fluorophore or a dye, a, in particular radioactive, isotope, a hapten, a particle, a plasmon resonance detectable substance, a ligand, in particular biotin, or a reducible or oxidizable substance. 25. The method of claim 24, wherein the fluorophore FAM (5 [6] -carboxyfluorescein), JOE (6-carboxy-4 ', 5'-dichloro- 2 ', 7'-dimethoxyfluorescein), ROX (5 [6] -carboxy-X-rhodamine) or TAMRA (carboxytetramethylrhodamine). 26. The method according to any one of the preceding claims, wherein the product with a double strand specific intercalator, especially SYBR Green. 27. The method according to any one of the preceding claims, wherein in step lit. e the marking substance ( 20 ) or the intercalator embedded in the product is detected. 28. The method according to any one of the preceding claims, wherein the nucleic acid sequence ( 16 ) to be detected is a DNA. 29. The method according to any one of claims 1-27, wherein the nucleic acid sequence ( 16 ) to be detected is an RNA, the compound ( 14 ) in step lit. b is additionally brought into contact with a reverse transcriptase and in step lit. c an additional reverse transcription is carried out. 30. The method according to claim 29, wherein in step lit. b an additional third primer specific for the nucleic acid sequence ( 16 ) to be detected is added, to which step lit. c can be added using the reverse transcriptase nu kleotide. 31. The method according to any one of the preceding claims, wherein the nucleic acid sequence ( 16 ) to be detected is double-stranded and before or during step lit. b is melted. 32. The method according to any one of the preceding claims, wherein the lit. c if necessary, in addition to the mo-modified compound (14) remaining not modify te compound (14) prior to step lit. d, in particular using silica particles, glass particles or a suitable filter, is removed. 33. The method according to any one of the preceding claims, wherein the amount of the binding sequence ( 26 ) immobilized on the carrier ( 22 ) is at least as large as the amount of the compound ( 14 ) provided overall. 34. The method according to any one of the preceding claims, wherein the first ( 10 ) and the second primer ( 18 ) are selected so that a hybrid formed from the first primer ( 10 ) and the nucleic acid sequence to be detected ( 16 ) has substantially the same melting temperature as a hybrid formed from the second primer ( 18 ) and the counter strand of the nucleic acid sequence ( 16 ) to be detected. 35. The method according to any one of the preceding claims, wherein the first ( 10 ) and the second primer ( 18 ), the identification sequence ( 12 ) and the binding sequence ( 26 ) are selected such that the melting point of a hybrid from the nucleic acid sequence to be detected ( 16 ) and the first primer ( 10 ) or a hybrid of the counter strand of the nucleic acid sequence ( 16 ) to be detected and the second primer ( 18 ) at least 5 ° C, in particular 10 ° C, above the melting point of a hybrid from the identification sequence ( 12 ) and the binding sequence ( 26 ) or from the opposite strand of the identification sequence ( 12 ) and the binding sequence ( 26 ). 36. A method of detection of multiple nucleic acid sequences (16) according to one of the preceding claims, wherein the Iden tifizierungssequenzen (12) and the binding sequences (26) are selected such that different frequencies from the Identifizierungsse (12) and specific to binding sequences (26) formed fish hybrids have essentially identical melting temperatures. 37. A method for the detection of several nucleic acid sequences ( 16 ) according to one of the preceding claims, wherein the identification sequences ( 12 ), the binding sequences ( 26 ) and the first ( 10 ), second ( 18 ) and optionally third primers are selected in such a way that they do not form unspecific hybrids under their respective stringent hybridization conditions. 38. A method for detecting a plurality of nucleic acid sequences ( 16 ) according to one of the preceding claims, wherein the first ( 10 ) and second primer ( 18 ) are selected such that different ones from the first primers ( 10 ) and the nucleic acid sequences ( 16 ) to be detected and the second primers ( 18 ) and the counterstrands of the nucleic acid sequences ( 16 ) to be detected specific hybrids have a substantially identical melting temperature. 39. A method for the detection of several nucleic acid sequences ( 16 ) according to any one of the preceding claims, wherein he most ( 10 ) and second primer ( 18 ) are selected so that in step lit. c Products are formed which are essentially of the same length. 40. A method for the detection of several nucleic acid sequences ( 16 ) according to one of the preceding claims, wherein the first primer ( 10 ) and / or the identification sequence ( 12 ) is / are specific for several of the nucleic acid sequences ( 16 ) to be detected and in each case one of the second primers ( 18 ) is specific for one of the nucleic acid sequences ( 16 ) to be detected and has a labeling substance ( 20 ) specific for this second primer ( 18 ), with step lit. e the specific marking substances ( 20 ) are additionally identified. 41. A method for the detection of several nucleic acid sequences ( 16 ) according to one of the preceding claims, wherein the steps lit. a, b and c with different nucleic acid sequences ( 16 ) to be detected separately from one another using distinguishable labeling substances ( 20 ) and / or intercalators and the steps lit. d and e are carried out together, with step lit. e the labeling substances ( 20 ) and / or intercalators are additionally identified. 42. Use of an identification sequence ( 12 ) in a method according to any one of claims 1-41, wherein the identification sequence ( 12 ) contains at least one agent preventing synthesis of a counter strand of the identification sequence ( 12 ) or wherein the identification sequence ( 12 ) nucleotides ( 13 ) and / or contains a nucleotide sequence which permits a degradation reaction. 43. Use of a first primer ( 10 ) having an activated coupling group or an identification sequence ( 12 ) having an activated coupling group in a method according to one of claims 1-41. 44. Use of a compound ( 14 ) formed from a first primer ( 10 ) and an identification sequence ( 12 ) specific for the first primer ( 10 ) in a method according to any one of claims 1-41, wherein in the compound ( 14 ) Means are provided which prevent synthesis of a counter strand to the identification sequence ( 12 ) during the reaction or wherein the identification sequence ( 12 ) contains nucleotides ( 13 ) and / or a nucleotide sequence which allows / allow a degradation reaction. 45. Use of a second primer ( 18 ) in a method according to any one of claims 1-41. 46. Use according to claim 45, wherein the second primer ( 18 ) has a marking substance ( 20 ). 47. Use of nucleotides provided with a labeling substance ( 20 ) in the reaction in a method according to any one of claims 1-41. 48. use of a double strand specific intercalator, especially SYBR Green, for marking the product in one Method according to one of claims 1-41.   49. Use of a carrier ( 22 ) having at least one immobilized binding sequence ( 26 ) in a method according to one of claims 1-41. 50. Use according to claim 49, wherein the binding sequence ( 26 ) is immobilized at a defined position of the carrier ( 22 ) or the carrier ( 22 ) is an identifiable carrier ( 22 ). 51. Use according to claim 49 or 50, wherein the carrier ( 22 ) consists of glass or plastic and / or is in the form of an identifiable particle, a membrane or a chip. 52. Use of a polymerase and / or a reverse Transcriptase in a method according to one of the claims 1-41. 53. Kit for carrying out a method according to one of claims 1-41, comprising:

• a) a binding sequence ( 26 ) immobilized on a support ( 22 ) and
• b) one (12) includes means to the binding sequence (26) at least partially complementary identifying sequence (12), wherein the Identi fizierungssequenz which approximately sequence according ei ner coupling a first primer (10) and the iden (12) in the reaction of a Synthesis of a counter strand to the identification sequence ( 12 ) prevented or
• c) an identification sequence ( 12 ) which itself or its counter strand which can be synthesized in the reaction is at least partially complementary to the binding sequence ( 26 ), the identification sequence ( 12 ) containing nucleotides ( 13 ) and / or a nucleotide sequence which contains a degradation action allow / permit.

54. Kit according to claim 53, wherein the identification sequence ( 12 ) has an activated coupling group for coupling the 5'-terminal region, in particular the 5'-end, of the first primer ( 10 ). 55. Kit according to claim 53, wherein a first primer ( 10 ) is included and either the identification sequence ( 12 ) or the first primer ( 10 ) is an activated coupling group for coupling the 5'-terminal region, in particular the 5'-end, of the first primer ( 10 ). 56. Kit for carrying out a method according to one of claims 1 to 41, comprising:

• a) a binding sequence ( 26 ) immobilized on a support ( 22 ) and
• b) a compound (14), formed from a facing for the nachzu nucleic acid sequence (16) specific first primer (10) and one for the first primer (10) specific to the binding sequence (26) at least partially complementary Iden tifizierungssequenz (12) wherein a compound is provided in the compound ( 14 ) which prevents a synthesis of a counter strand to the identification sequence ( 12 ) during the reaction or
• c) a compound (14), formed from a facing for the nachzu nucleic acid sequence (16) specific first primer (10) and are specific to the first primer (10) Iden tifizierungssequenz (12), the identifying sequence (12) nucleotides (13 ) and / or contains a nucleotide sequence which allows / allow a degradation reaction and wherein the identification sequence ( 12 ) or its counter strand which can be synthesized during the reaction is at least partially complementary to the binding sequence ( 26 ).

57. Kit according to one of claims 53-56, wherein the binding sequence ( 26 ) is immobilized at a defined position on the carrier ( 22 ) or the carrier ( 22 ) is an identifiable carrier ( 22 ). 58. Kit according to one of claims 53-57, wherein the carrier ( 22 ) consists of glass or plastic and / or is in the form of an identifiable particle, a membrane or a chip. 59. Kit according to one of claims 53-58, wherein the agent comprises at least one nucleotide analog, RNA, PNA or an interruption in the sequence of the phosphodiester bonds connecting the nucleotides in the compound ( 14 ), in particular by means of a polyethylene glycol linker, a peptide bond or a disulfide bond. 60. Kit according to one of claims 53-59, wherein a polymer rase and possibly a reverse transcriptase is / are included. 61. Kit according to one of claims 53-60, a second primer ( 18 ) having, in particular, a marking substance ( 20 ) being contained. 62. Kit according to one of claims 53-61, wherein nucleotides provided with a labeling substance ( 20 ) are contained. 63. Kit according to one of claims 53-62, wherein a double strand-specific intercalator, in particular SYBR Green, is included.