DE4041608A1 - Sensitive and selective nucleic acid detection - Google Patents

Abstract

Specific detection of a nucleic acid (A) in a sample comprises (a) reacting the sample with 1 or more labelled mononucleotide triphosphates (I) and one or more enzymes which catalyse prepn. of labelled nucleic acid (B) contg. (I); (b) recting the sample with a nucleic acid probe (C) which is adequately complementary to (B) and (c) detecting the hybrid (D) formed between (B) and (C). The new features are that (1) (C) contains at least one immobilisable gp. (2) after step (a) a non-thermal denaturation is carried out; (3) (D) is contacted with a solid phase which can bind specifically to immunoilisable probe (c); (4) the liq. and solid phases are sepd. and (5) the label is detected on the solid phase. Step (a) is carried out in presence of a specific initiator and the enzymes used produce a (B) essentially complementary to (A). The initiator is pref. a primer (P1) complementary to a a part of (A) and is extended in step (a). A second primer (P2) complementary to part of (B) can also be included and after extension reaction the prods. are sepd., reacted again with P1/P2 and used as template for extension of the corresp. primer.

Description

The subject of the patent application is a method for specific detection of a nucleic acid or a part from that.

The detection of nucleic acids in clinical diagnostics and molecular biology has recently compared to classic immunoassays are becoming increasingly important. This is connected with the fact that progress towards the Research into the nucleotide sequence of nucleic acids of different origins.

Because the information contained in the nucleotide sequence is very are differentiated, nucleic acid diagnostics in particular advantageously used to distinguish the smallest features will. This is important, for example, when detecting Mutations and differences in artificially created sequences. Through the introduction of amplification processes, the Sensitivity of nucleic acid detection increased considerably will.

Such a method is described, for example, in EP-A-02 00 362 described. The amplification effect is essentially based on the fact that from a so-called target nucleic acid as template using a primer and mononucleotide triphosphates an extension product of the primer is formed which is either verified or again as a template Nucleic acid can be used. It can in that  Extension product also incorporated a detectable nucleotide will. The resulting extension product can be detected electrophoretically.

However, this method of detection is cumbersome and time-consuming electrophoresis step disadvantageous.

The unmarked extension product can be according to EP-A- 02 01 184 also detectable by hybridization with a labeled nucleic acid probe can be detected. The Separation of the hybrid from extension product and probe from unreacted probe is however with that described there Non-specific interaction procedures either inefficient or associated with many washing steps, which reduce sensitivity.

WO 89/11 546 proposes a method in which amplified, solid phase bound nucleic acids with a Primers and labeled mononucleotides are implemented, whereby the labeled nucleic acids formed are detected will. This method has the disadvantage that all significant reactions take place on the solid phase, whereby the reaction speed is impaired. Also in the EP-A-03 24 474 proposes such a method in which incorporated labeled mononucleotides and labeled them Nucleic acids with the nucleic acid to be detected complementary immobilized nucleic acids captured will. About the method of denaturing the amplified Nucleic acids and the hybridization conditions are nothing testified. Another disadvantage of this method is that the Production of efficiently solid-phase bound nucleic acids is complex.  

EP-A-03 57 011 describes a method in which two primers are used in the extension reaction, from one of which is demonstrably marked and the other for binding a solid phase is suitable. This procedure has the Disadvantage that it is more elaborately marked Oligonucleotides from those containing these oligonucleotides To separate extension products. If no partition is made because of competitive reactions reduced sensitivity to be expected.

EP-A-02 97 379 and EP-A-03 48 529 describe a method described in which an immobilized or immobilizable Primer using the target nucleic acid as a template immobilized with a detectable mononucleotide and at the same time detectably labeled nucleic acid extended becomes. This method has u. a. the disadvantage that the Specificity of the proof is not very high. That also in Process described in EP-A-02 97 379, in which only one immobilized or immobilizable primer is used, whereupon the extension products with a marked Implemented oligonucleotide has the EP-A-03 57 011 described disadvantage of the difficult severability of the Oligonucleotide.

WO 89/09 281 describes a method in which the both primers have the same chemical group that once used for immobilization and the other for detection becomes. This exacerbates the above disadvantage of bad Detachability still.

In Proc. Natl. Acad. Sci. USA, Vol. 86, pp 6230-6234 also described a method in which a detectable marked primer is extended. Evidence is provided after Binding the extension products to a catch sample. Here too  is not the separation of the demonstrably labeled primer possible without additional steps, and it therefore interferes with the Detection reaction.

It was therefore an object of the invention the disadvantages of the method to avoid the prior art and in particular a To provide nucleic acid detection method, which is high Specificity or selectivity with a low background signal connects.

The invention relates to a method for specific Detection of a nucleic acid in a sample, the following Steps include:

• a) Reaction of the sample with one or more labeled Mononucleoside triphosphates and one or more Enzymes that are used to produce a labeled Nucleic acid B catalyze this nucleotide contains
• b) reaction of the sample with a nucleic acid probe C, which are sufficiently complementary to nucleic acid B is
• c) detection of the from labeled nucleic acid B and Nucleic acid probe C formed nucleic acid hybrid D,
• d) wherein the nucleic acid probe is at least one contains immobilizable group,
• e) the reaction mixture after step a) not one undergoes thermal denaturation,  
• f) the nucleic acid hybrid D with a solid phase, which the immobilizable nucleic acid probe C can bind specifically, is brought into contact,
• g) the liquid is separated from the solid phase and
• h) the detectable group bound to the solid phase is proven.

The method according to the invention is a special embodiment of the so-called hybridization tests, the basic features of which are known to those skilled in the field of Nucleic acid diagnostics are known. So far experimental Details that are not detailed below will do so full content on "Nucleic acid hybridization", publisher B.D. Hames and S.J. Higgins, IRL Press, 1986, in Chapters 1 (Hybridization Strategy), 3 (Quantitative Analysis of Solution Hybridization) and 4 (quantitative filter hybridization), Current Protocols in Molecular Biology, Edt. F.M. Ausubel et al., J. Wiley and Son, 1987, 2.9.1.-2.9.10 and Molecular Cloning, Edt. J. Sambrook et al., CSH, 1989, 9.4.7.- 9.5.8. Referred. These include in particular the known ones Methods for the production of labeled nucleoside triphosphates, as also described in EP-A-03 29 474, the chemical synthesis of modified and unmodified Oligonucleotides using nucleic acids Restriction enzymes, the selection of hybridization conditions by which specificity can be achieved the degree of homology between those to be hybridized Nucleic acids, the GC content and length of which depend, and with the formation of nucleic acids from nucleoside triphosphates With the help of polymerases, optionally using so-called primers.  

A label in the sense of the present invention consists of a directly or indirectly detectable group L. Directly detectable groups are, for example, radioactive ( ³² P), colored or fluorescent groups or metal atoms. Indirectly detectable groups are, for example, immunologically or enzymatically active compounds such as antibodies, antigens, haptens or enzymes or enzymatically active partial enzymes. These are detected in a subsequent reaction or reaction sequence. Haptens are particularly preferred since nucleoside triphosphates labeled with them can generally be used particularly well as substrates for polymerases and a subsequent reaction with a labeled antibody against the hapten or the haptenized nucleoside can easily be carried out. Such nucleoside triphosphates are, for example, bromine nucleoside triphosphates or digoxigenin, digoxin or fluorescein-coupled nucleoside triphosphates. The steroids mentioned in EP-A-03 24 474 and their detection have proven to be particularly suitable. For their incorporation into nucleic acids, reference is hereby made to EP-A-03 24 474.

Nucleoside triphosphates (NTP) are ribo (rNTP) - or Deoxyribonucleoside triphosphates (dNTP).

Under a target nucleic acid is a nucleic acid understood the aim of the proof and starting material of the is the inventive method.

A template nucleic acid A is a nucleic acid to which a essentially complementary nucleic acid strand newly formed becomes. With regard to the sequence information, the template Nucleic acid as a template and contains the sequence formation that in reaction a) is rewritten into nucleic acid B. The Nucleic acid A is either the target nucleic acid or a  nucleic acid derived therefrom. For example, it can be a Be part of the target nucleic acid or part of the target Contain nucleic acid among other parts, for example highly complex nucleic acids. It can also be part of the contain target nucleic acid complementary strand.

Denaturation means separation of Nucleic acid double strands in single strands. The specialist a large number of variants are available.

A specific proof is a procedure understood by which, if desired, selectively determined Nucleic acids also in the presence of other nucleic acids can be demonstrated. However, it is also possible to have several Partial nucleic acids or a group of nucleic acids matching or similar nucleotide sequence or more Sections of one nucleic acid in the presence of another Detect nucleic acids. For the detection of double-stranded Nucleic acids can be any of the two complementary strands be included.

Under an essentially complementary nucleic acid or Nucleic acid sequences become nucleic acids or sequences understood that with the corresponding nucleic acid can hybridize, the nucleotide sequence in the hybridizing area either exactly complementary to that other nucleic acid or in a few bases from the distinguishes exactly complementary nucleic acid. The specificity depends on the degree of complementarity as well as the hybridization conditions.

Liquid phases are the most common Nucleic acid detections used aqueous phases with dissolved organic or inorganic ingredients, e.g. B.  Hybridization buffer, excess nucleotides, others undetectable nucleic acids, proteins etc.

The method according to the invention serves to detect a Nucleic acid. Nucleic acids are nucleic acids to understand any origin, for example nucleic acids viroid, viral, bacterial or cellular origin. they can be fixed in solution, suspension, but also on solids or in cell-containing media, cell smears, fixed cells, Tissue sections or fixed organisms are present. Prefers the nucleic acids are in solution. The reaction sequence is usually started by making the investigating nucleic acid with appropriate reagents. Changes in pH (alkaline), heat, Repetition of extreme temperature changes (Freeze / thaw), change in physiological Growth conditions (osmotic pressure), exposure to Detergents, chaotropic salts or enzymes (e.g. proteases, Lipases), alone or in combination to release the Contribute nucleic acids. Since the method according to the invention is very sensitive and selective, even small ones can Amounts of nucleic acids in the presence of other substances, for example proteins, cells, cell fragments, but also not detectable nucleic acids. A Purification of samples can therefore be omitted if it is ensured that the nucleic acids to be detected for the reagents used are sufficiently accessible.

The nucleic acids can also be pretreated. To the known pretreatments include in particular the Fragmentation, for example using restriction enzymes or cDNA synthesis from RNA. The method according to the invention can fully unfold its advantages, it has proven to be useful  proven if the nucleic acid is at least 40 bp in size having.

Furthermore, the nucleic acid is preferably the product of an upstream specific or non-specific nucleic acid amplification. Such nucleic acid amplification processes are, for example, from
EP-A-02 01 184, EP-A-02 72 098, DE-A-37 26 934,
EP-A-02 37 362, WO 88/10 315, WO 90/01 069, WO 87/06 270,
EP-A-03 00 796, EP-A-03 10 229, WO 89/09 835,
EP-A-03 70 694, EP-A-03 56 021, EP-A-03 73 960,
EP-A-03 79 369, WO 89/12 696 or EP-A-03 61 983
known. However, the nucleic acid can also be a nucleic acid produced by cloning and in vivo propagation.

The (target) nucleic acids to be detected can be used directly as template nucleic acids A are used in reaction a) if the conditions required for the chosen enzyme system fulfill. For some enzymes, this means that it is single-stranded nucleic acids, others are required that they have recognition sites or promoters for the Enzyme system include.

If this is not the case, the target nucleic acids must be in a (reaction a) upstream step in such template nucleic acids A are transferred.

A can be a ribonucleic acid or a Act deoxyribonucleic acid. Particularly preferred as Nucleic acid A are deoxyribonucleic acids.  

The enzyme E in the sense of the invention is an enzyme or Enzyme system that supports the templated synthesis of Catalyzed nucleic acids from mononucleoside triphosphates. Preferred enzymes are polymerases and transcriptases, which Link mononucleotides. Such enzymes are known to the expert.

In a first step, the template nucleic acid A a labeled nucleic acid B is produced. This can be due to basically any way happen as long as only that specific information of the nucleotide sequence of nucleic acid A or part of it remains largely intact.

One method is, for example, the exchange of individuals Nucleotides of nucleic acid A against labeled nucleotides, for example by Nick repair (J. Mol. Biol. 113, 237 (1977)). The enzyme E in this reaction is E. coli DNA Polymerase or Kornberg enzyme. However, this procedure is not too advantageous since the marking is relatively unspecific is introduced, d. H. in addition to nucleic acid A all others in nucleic acids present in the sample are marked. It is suitable therefore for reaction a) especially if, as above described in an upstream propagation reaction nucleic acids to be detected were specifically amplified.

In another method, which is also the one mentioned Disadvantage, the nucleic acid A with incorporation of labeled nucleotides extended (tailing). Other procedures of this kind are photo marking and random priming.

In the previously described steps for producing the In reaction A, nucleic acid B is the target Nucleic acid used. This can be single or double-stranded are available.  

The target nucleic acids listed below preferred variants must be before or during the Reaction a) be made single-stranded. A possibly necessary strand separation can be achieved by treatment with alkali, but also thermally, enzymatically or using chaotropic salts happen.

The use of reactions a) which are dependent on the presence of a specific starter is particularly preferred because of the increased specificity. Such specific starters have the effect that the enzyme acts only on the nucleic acid which has bound this starter. The starter is preferably a so-called specific primer P 1 , a promoter or a replication initiation region.

Specific primers P 1 are in particular oligonucleotides or modified oligonucleotides which have a nucleotide sequence essentially complementary to nucleic acid A. Modified oligonucleotides are, for example, oligonucleotides which contain a dideoxynucleotide at the 3'-end. Such oligonucleotides can be produced enzymatically. The target nucleic acid can be used directly as template nucleic acid A.

The use of such primers P 1 therefore presupposes that at least part of the sequence of the nucleic acid is known. The sequence of the primer is also chosen so that it is shorter at one of its ends, preferably at the 3'-end, than the nucleic acid. This therefore has a single-strand protruding portion beyond the 3'-end of the primer.

One or more specific primers can be used in the reaction step for each nucleic acid single strand to be detected. In the case of several primers, the regions on the nucleic acid with which the primers can hybridize preferably do not overlap, and particularly preferably single-stranded regions of nucleic acid A lie between these regions. In addition to a part which is essentially complementary to template nucleic acid A, the primer can , preferably at the 5 'end, also contain a nucleotide sequence S 1 which cannot hybridize with the nucleic acid, in particular the region adjoining the complementary region.

This sequence S 1 can for example be single or double-stranded and also contain a recognition sequence for an enzyme. This can e.g. B. be a restriction site. With regard to effective priming, the primer can also contain, for example, a protein which is recognized by an enzyme E, preferably a polymerase.

In order for reaction a) to take place, the nucleic acid A and the primer P 1 must first be separated in the complementary region from any complementary strands which may be present. This separation can take place according to known methods, for example thermally or non-thermally. Non-thermal denaturation is preferred. Reaction a) is then started under conditions in which A and P 1 can hybridize with one another.

When using a primer as a starter, the sample also added an enzyme E, which in a primer-dependent reaction to A complementary nucleic acid can synthesize. These include in particular polymerases.  

Their substrate specificity depends on nucleic acid A. If A is RNA, RNA-dependent DNA Polymerases, such as reverse transcriptase (e.g. from AMV or M- MuLV) in question. If A is DNS, then DNS-dependent DNS Polymerases, such as the Klenow enzyme of Taq DNA polymerase prefers.

Deoxyribonucleoside is also added to the sample triphosphates (dNTP), at least one of which is labeled.

The product of the polymerase reaction is a labeled deoxyribonucleic acid B, in which the primer P 1 and the labeled deoxyribonucleoside triphosphate are incorporated. This nucleic acid B is the same size or smaller than the template, but has an area which is at least in part substantially complementary to the template.

This nucleic acid B can now be used directly in step b). However, it is advantageously subjected to a reaction analogous to step a) as template nucleic acid. For this purpose, a primer P 2 is added to the sample, which is essentially complementary to part of nucleic acid B, preferably part of the region newly formed from dNTPs. The primer pair P 1 and P 2 preferably meet the conditions described in EP-A-02 01 184. P 1 and P 2 are preferably added simultaneously to template A.

In order to achieve an even higher sensitivity, it is possible to carry out reaction a) a number of times, preferably 1-60, particularly preferably 20-60 times, the products of the reaction being used again in reaction a). Theoretically, this results in an almost exponential increase in labeled nucleic acids. Both strands of nucleic acid are formed, the length being determined by the non-extendable ends of the primers P 1 and P 2 . Analogously to EP-A-02 01 184, it is also possible to use so-called nested primers in the later runs of reaction a), the sequence of which is selected such that the nucleic acids formed are smaller than those initially formed. Before each run of reaction a), it is advantageous to separate the double strands of A and B formed in reaction a). This can be done thermally or not thermally. Primers P 1 and P 2 must be present again.

The starter can also be a promoter. A promoter is one Nucleotide sequence recognized by an RNA polymerase and this causes one to follow the promoter Nucleotide sequence complementary nucleic acid strand B to synthesize. In addition to the unmarked NTPs, the labeled NTP in the nucleic acid strand formed built-in.

Suitable promoters are known, for example RNA polymerase Binding sites from bacterial phages such as T3, T7 or SP6 (Melton et al .: NAR 12, 1984, 7035-56; Pfeiffer & Gilbert: protein Sequences and DNA Analysis I, 1988, 269-280; Uhlenbeck et al .: Nature 328, 1987, 596-600).

In a preferred embodiment of the detection method, the promoter is part of a specific primer for producing the template nucleic acid A from the target nucleic acid. This primer has a nucleotide sequence S 1 , which is essentially complementary to part of the target nucleic acid, and a nucleotide sequence S 2 , which is recognized by an RNA polymerase and contains at least one promoter sequence. In a first reaction, a nucleic acid strand A 1 which is at least partially complementary to the target nucleic acid and which contains the primer with the promoter is formed with the primer and a DNA polymerase and dNTPs which depend on the type of target nucleic acid. If not already connected to the primer, the newly formed nucleic acid piece is added by adding another enzyme, preferably a ligase, e.g. BEcoli DNA ligase covalently linked to the primer. The ligase can also be thermally stable. A 1 is preferably shorter than the target nucleic acid.

In this transcription reaction, a second primer P 3 which is complementary to the target nucleic acid strand is preferably used and the gap between the two primers is closed, for example by a gap filling reaction. The use of marked dNTPs is not yet necessary here, since this measure has an effect in the method according to the invention in only a slight amplification of the measurement signal, but is possible.

If the target nucleic acid is RNA, it is preferably selectively degraded in a subsequent step. Known methods are suitable for this, for example treatment with alkali or with RNAsen. A nucleic acid strand A 2 complementary to A 1 is then formed. For this purpose, a primer P 2 is added to the reaction mixture, which is complementary to part, preferably a newly formed part of strand A 1 . The primer P 2 preferably also contains the promoter sequence S 2 . As described above for A 1 , this is extended to A 2 . Such process steps are e.g. B. in EP-A-03 29 822, DE-A-37 26 934, WO 88/10 315, WO 87/06 270, EP-A-03 10 229 and EP-A-03 73 960, which is why full reference is made here to these disclosures. Reference is made to this disclosure in particular with regard to details that are helpful and necessary for reverse transcription of RNA or transcription of DNA.

In this embodiment, the sample particularly preferably also contains the strand which is complementary to the target nucleic acid, P 1 and P 2 being extended at the same time.

In the embodiment mentioned, the sample pretreated in this way is then used, according to the invention, with a promoter-specifically controlled DNA-dependent RNA polymerase. Such polymerases are e.g. B. T3, T7 or SP6 RNA polymerase. With their help, labeled nucleic acids B are formed from NTPs and the labeled NTP. The double-stranded nucleic acid A 1 / A 2 serves as a template nucleic acid, preferably several times.

Preferred NTPs are ribonucleotide triphosphates. Because with this Variant of the reaction a) single-stranded nucleic acids B is a denaturation for strand separation not absolutely necessary, but possible.

A preferred embodiment is the procedure according to DE-A- 40 10 465, but using demonstrably marked Ribonucleotide triphosphates.

The starter is preferably a replication initiation region (RIR). A replication initiation region is, for example, a nucleotide sequence that is recognized by a replication enzyme or - enzyme complex, for example a DNA-dependent DNA polymerase, as by ⌀29 (P 2 , P 3 ). It is particularly preferred that the RIR is part of a specific primer. This primer has a nucleotide sequence S 1 which is essentially complementary to part of the target nucleic acid and then a sequence S 2 which has a recognition site, preferably proteins, for a replication complex. Such a primer is called adapter Ad in the following. The reaction is initiated by reacting the target nucleic acid, which is actually the species to be detected, with Ad under hybridization conditions. The use of two adapters Ad 1 and Ad 2 per target nucleic acid single strand is particularly preferred, the target-specific sequences being able to hybridize with spatially separated sequences of the single-stranded target nucleic acid and the target-specific sequences of the adapter on the target nucleic acid being directed towards one another.

The single-stranded gap between the adapters is defined by a gap-filling reaction (Maniatis et al., Molecular Cloning, 1982, CSH). The use of marked NTPs is not required, but possible. This creates a Nucleic acid duplex, which is one strand of the target Nucleic acid and a strand of the newly formed nucleic acid A contains. This contains at least one, preferably two Replication initiation region, which is particularly preferred in opposite direction of replication are arranged.

From this double strand can now according to the invention through replication without further addition of adapters Use of marked and unmarked Nucleoside triphosphates in reaction a) a labeled one Nucleic acid B can be produced. This in turn contains at least one, preferably 2, replication initiation regions and can therefore again in reaction a) as a template nucleic acid be used. A considerable increase in B reached. In the event that both adapters each have an RIR have, or two mutually complementary nucleic acids A  can be used in reaction a), two to each other complementary nucleic acids B arise. These must before step b) are separated from one another by denaturation.

After step a), the reaction mixture is subjected to a non-thermal denaturation (reaction e)) in the process according to the invention. Even if step a) is carried out several times, the non-thermal denaturation takes place directly before step b). In particular, nucleic acid double strands are separated from one another. Non-thermal denaturing means that the temperature for denaturing does not take place above 50 ° C., preferably not above 37 ° C., preferably in a range between 22 and 37 ° C. During denaturation, any double strands of nucleic acid present are separated from one another, so that nucleic acid B is present as a single strand. This can be done, for example, by changing the stringency, but also by enzymes, for example helicase, recA protein, E. coli ssb protein, Gen-32 protein, and by alkali or by adding double-strand-stabilizing chemicals, such as. B. organic substances such as urea, formamide (concentration <70%, NAR 1977, Vol. 4, 5, 1539-1553) or chaotropic salts. Chaotropic salts are described in J. Biol. Chem. 1966, 241/17, 4030-4042 J. Amer. Chem. Soc. USA 1962, 84/8, 1329-1338 or Anal. Biochem. 129, 357-364 (1983). NaI, guanidine thiocyanate or NaClO ₄ are particularly preferred for the purposes of the invention. The concentration of these salts required for denaturation can be determined by simple experiments. However, it is z. B. for NaJ preferably about 12.2 mol / l, for guanidinium salts about 4 mol / l and for NaClO ₄ about 7 mol / l.

The alkaline has been found to be particularly advantageous Denaturation proven. For this, the reaction mixture is made Reaction a) to a pH in the range from 10 to 14, preferably from 12 to 14. This can be done, for example, by adding a solution of NaOH, KOH in water.

The temperature of the mixture is during the incubation of 1 to 30 min, preferably 5 to 10 min, in a range of 17 to 50 ° C, preferably 22 to 37 ° C.

In the following reaction step b) nucleic acid B with the Probe C reacted so that it together Form nucleic acid hybrid D.

In particular, oligo- or poly- Nucleotides with a length of 6 to 5000, preferably 15 to 2000 in question. Probe C can be a plasmid Nucleic acid fragment or an oligonucleotide. It can are RNA or DNS. Nucleic acid probe C is in excess to the expected amount of nucleic acid B to the Reaction mixture advantageously in the form of an aqueous solution admitted. Probe C has a nucleotide sequence which is in the is essentially complementary to B, and which is specific for B. is, so not or only to a very small extent in the Sample existing or newly formed nucleic acids hybridized, which should not be detected. Through the Combination of the use of specific primers and the Hybridization with a specific probe will The method according to the invention is particularly selective.  

C may be a double-stranded nucleic acid, one strand of which C 1 is complementary to a portion of B. The other strand C 2 of the nucleic acid probe is preferably complementary to other nucleic acids B, in particular to those which arise when reaction a) is carried out with B as the template nucleic acid. C can be denatured separately from B. However, it is preferred to denature C together with B. This applies in particular to alkaline denaturation. For this purpose, C is preferably added before the alkalization of the reaction mixture from a). After an incubation period, the mixture is brought to a pH of 5.0 to 8.5, preferably 7-8, whereupon the nucleic acid hybrids D form B and C 1 or C 2 .

The preferred case is that C is a single-stranded nucleic acid probe C 1 and the strand C 2 complementary to C 1 is not added. It is then also possible to add C 1 before denaturing B. In the case of the alkaline denaturation of B, however, it is particularly preferred to first incubate only B in an alkaline manner and then to add an acidic solution to the probe C 1 . The pH of the acidic solution of C 1 is in the range from 3.0 to 6.5, preferably from 4.5 to 6.0, without the sensor being noticeably unstable. After addition to the hybridization solution, the weakly acidic solution should be sufficient to set a pH of 5.0 to 8.0. The solution can also be buffered in this area and contain other reagents useful for hybridization, such as. B. SSC, formamide or blocking reagents for undetectable nucleic acids. The pH of the mixture after addition of C 1 should also be in the range from 5.0 to 8.5. The pH can be finely regulated, for example, by the amount of hybridization solution and its pH.

Acids used for neutralization or as part of the Hybridization solution can be used for example hydrochloric acid or formic acid; are preferred however, acetic acid or phosphoric acid. The pH of the Hybridization solution before addition to the sample is between 3.0 and 6.5. The concentration is based on the example of acetic acid preferably between 0.05 and 0.5 mol / l, particularly preferably 0.1 and 0.2 mol / l.

Single-stranded nucleic acid probes C 1 can be produced, for example, by chemical nucleic acid synthesis according to DE-A-39 16 871 or also according to EP-B-01 84 056.

This embodiment has the advantage that by using the Probe solution to neutralize the sample solution Pipetting step can be saved. Besides, it did proved that the addition of a concentrated acid to the alkali-treated sample with a high protein content Clumping leads, which is why the proposed approach is particularly advantageous.

If denaturation is carried out using step b) double strand destabilizing substances was made, conditions for hybridization with probe C be adjusted by diluting the mixture.

Probe C contains one or more for each strand of nucleic acid Immobilizing groups (immobilisable) I.

Groups I capable of immobilization are, for example chemical groups that are covalent, for example via a chemical or photoreaction bound to a solid phase can be, or groups or parts of molecules that over group-specific interactions from another molecule  or part of the molecule can be recognized and bound. Such Groups are therefore z. B. haptens, antigens and antibodies, Nucleotide sequences, receptors, regulatory sequences, Glycoproteins, for example lectins, or also Binding partner of binding proteins, such as biotin or Iminobiotin. Vitamins and haptens are particularly preferred preferred are biotin, fluorescein or steroids, such as Digoxigenin or digoxin. It is important for the invention that in each hybrid D the immobilizable group of the probe itself differs from the detectable group of nucleic acid B.

The mixture which contains the nucleic acid hybrid D if the nucleic acid to be detected was present in the sample then brought into contact with a solid phase which the hybrid D via the immobilizable groups of Can specifically bind nucleic acid probe C.

The type of solid phase depends on that for immobilization enabling group I. It preferably has one immobilizing group R, which has a binding interaction can enter into with I. Is the immobilizable group for example a hapten, then a solid phase can be used that have antibodies against this hapten on their surface having. Is the immobilizable group a vitamin like e.g. B. biotin, then the solid phase binding proteins, such as Avidin or streptavidin immobilized included. Especially preferred residues I and R are biotin and streptavidin. The Immobilization via a group on the modified Nucleic acid is particularly beneficial because it is milder Conditions can take place as for example Hybridization reactions.  

Preference is given to immobilizing the nucleic acids formed the reaction mixture after formation of the nucleic acid hybrids D in a vessel filled with the surface of the immobilizable group can react. Preferably the Hybridization reaction with the probe simultaneously with the Immobilization instead. The vessel can be, for example Cuvette, a tube or a microtiter plate. It is however also possible a solid phase in the form of a porous Material, such as a membrane, a fabric or a fleece, to use, to which the reaction mixture is applied. Likewise, the use of beads, so-called beads, or Latex particles possible. The solid phase should at least so many binding sites for the immobilizable group of Have probes like nucleic acid hybrids D and thus nucleic acids B are present.

The preparation of a preferred solid phase is described in EP-A- 03 44 578, to which full reference is made becomes.

After an incubation period during which the immobilization reaction takes place, the liquid phase from the vessel, the porous material or the pelleted beads removed. The Solid phase can then with a suitable buffer be washed since the binding of the hybrid D to the solid phase is very efficient. The method according to the invention allows the use of particularly fewer washing steps, since the difficult to separate probes in contrast to in the prior art detectable probes used are not necessarily complete must be removed, or leads to comparatively small Background signals.  

The amount of modified bound to the solid phase Nucleic acids can be determined in a manner known in principle the steps to be carried out according to Art the detectable group. With directly detectable Groups, for example fluorescent labels, will change the amount Label determined fluorometrically. The detectable group is a Hapten, the modified nucleic acid is preferred with a labeled antibody against the hapten, such as described analogously in EP-A-03 24 474. The marking can for example an enzyme label, such as β-galactosidase, alkaline phosphatase or peroxidase. In the case of Enzyme labeling is the amount of nucleic acid over the most photometric, chemiluminometric or fluorometric Monitoring a reaction of the enzyme with a chromogenic, chemiluminogenic or fluorogenic substrate measured. It is however, electrochemical monitoring of the reaction is also possible, if a redox enzyme is used as a label, or the Tracking a pH change using a pH electrode. The measurement signal is a measure of the amount of originally existing target nucleic acid.

The detection of nucleic acid can be both qualitative and be managed quantitatively. In the case of a quantitative Evaluation it turned out to be useful known at least one comparison test with a sample Perform nucleic acid content. The creation of a Calibration curve is possible and recommended.

In one embodiment of the method using PCR, oligonucleotides are added to the sample as primers P 1 and P 2 . P 1 is complementary to part of the nucleic acid single strand A, which represents the target and at the same time the template nucleic acid. P 2 is homologous to a part of A removed therefrom. The mixture is now treated as described in EP-A-02 01 184, but in addition to the unmodified deoxymononucleotide triphosphates, a digoxigenin-labeled or fluorescein-labeled deoxymononucleotide triphosphate is also used. 20-30 amplification cycles are preferably carried out. Then double-stranded biotin-labeled probe C and sodium hydroxide solution are added to a pH of 10-14. The mixture is incubated at about 37 ° C. and then a solution of hybridization aids is added at about pH 5, so that a pH of about 7-8.5 results. The mixture is transferred to a streptavidin-coated vessel and incubated again. The solution is removed and the vessel is washed. A conjugate of antibodies against digoxigenin and an enzyme is added and incubated again. After removing the solution and washing the vessel, it is reacted with a chromogenic substrate of the enzyme and the color formation is observed.

A preferred embodiment is a variant of the replication method described in DE-A-39 29 030. Reference is hereby made in full to the disclosure of DE-A-39 29 030. To carry out the method, the sample which contains the single-stranded target nucleic acid is provided with two adapters which are complementary to the same strand and whose ends hybridizing with A are directed towards one another and whose non-hybridizing ends contain a replication initiation sequence for ⌀29 polymerase , offset. Under hybridization conditions, the gap between the adapters is closed by gap-filling using DNA polymerase or reverse transcriptase (as enzyme E 1 ) and ligase reaction. The deoxy mononucleoside triphosphates used here can be unmodified, but preferably one of them is a digoxigenin-labeled monodeoxyribonucleoside triphosphate. To carry out the replication, the replication system of ⌀29 (P 2 , P 3 and ATP) and, in addition to unmodified mononucleoside triphosphates, if not already used in the gap-filling reaction, a digoxigenin- or fluorescein-labeled monodeoxyribonucleoside triphosphate are added. The detectably labeled nucleic acids B formed by replication serve again as template nucleic acids for the formation of further nucleic acids B without further addition of adapters. Therefore, theoretically there is an exponential increase in the number of detectably labeled nucleic acids B over time. As soon as sufficient nucleic acids have been formed, the Mixture subjected to a non-thermal denaturation step and mixed with a preferably single-stranded biotin-labeled probe C 1 in a solution with hybridizing reagents at pH 3.0-6.5 up to a pH of 5.0-8.5, preferably 7.0-8.0. By introducing the non-thermal denaturation step, it is possible to carry out the entire detection method in a temperature range between 17 and 50 ° C. and preferably at a single temperature. The mixture is transferred to a streptavidin-coated vessel and incubated. After sucking off the liquid and washing the vessel, a solution of a conjugate of an enzyme-labeled antibody against digoxigenin or fluorescein is added and incubated again. After the solution has been suctioned off and washed, a solution of a chromogenic substrate is added and the color formation is observed.

In the prior art processes have always been detectably labeled probes are used. A complete Separation of the non-hybridizing probes was for the Accuracy of the measurement result required, but relative complex.  

The method according to the invention circumvents this disadvantage by instead of hybridizing labeled probes and their Determining the presence of built-in marked Mononucleotides measured and as a measure of the presence or the amount of nucleic acids to be detected is taken. The Removal of unincorporated labeled mononucleotides is after the present method particularly simply and completely achieve since they do not affect the nucleic acids or the Surface bound. An excess of immobilizable However, probe C does not interfere with the determination, since the immobilizable groups from probe C not as a label are used and therefore do not contribute to the measurement result. In particular, the binding capacity of the solid phase is better calculable than when installing immobilizable NTPs.

The method according to the invention is therefore very sensitive and selective, it can also be done in a short time.

The method according to the invention is suitable for the detection of Nucleic acids as such, from nucleic acid-containing organisms e.g. B. bacteria, viruses and cells and for the detection of Nucleic acid changes, such as mutations or Chromosome translocations and for localization and for Expression level of genes. Both ribonucleic acids as well Deoxyribonucleic acids can be detected. Also the Identification of point differences in nucleic acids is possible when comparing the results of experiments are used in which once a primer in reaction a) is complementary to the nucleic acid A at a 3'-end is, on the other hand, a primer is used, which is attached to its 3'- End just isn't complementary to nucleic acid A, however hybridized with her anyway. An extension of the primer and thus formation of nucleic acid B is only in the first case  to be possible. The selection of such primers is, for example, in PNAS 86, 2757 (1980).

Fig. 1 shows schematically the sequence of an embodiment using a primer elongation (using only one primer P 1).

Fig. 2 shows schematically the sequence of an embodiment using the PCR-principle with two primers, wherein the nucleic acids B initially formed are re-used as a template nucleic acid.

Fig. 3 shows schematically the sequence of the preferred embodiment using a Replikationsamplifikation.

FIG. 4 shows in curve I the result of an experiment according to FIG. 1. The absorbance measured in the solution against the amount of nucleic acid A to be detected is plotted against one another; Curve II shows the result without the presence of the nucleic acid to be detected.

Fig. 5 shows a calibration curve 1 for a determination of HBV-DNA by PCR, and a single stranded biotin-labeled oligonucleotide according to Example 2. Curve 2 shows the blank value (without addition of organic Oli 25).

Fig. 6 shows a calibration curve 3 for the determination of HBV in a method according to Example 3. Curve 4 shows the blank value (without addition of organic Oli 25).

Reference numerals

A template nucleic acid
A 1 counter strand of A
P 1 primer complementary to A.
E enzyme / enzyme complex
E 1 DNA polymerase or reverse transcriptase
L detectable groups (marking)
B product of reaction a)
C nucleic acid probe
I immobilizable group
D nucleic acid hybrid from B and C
R immobilizing group
F solid phase
S 1 sequence complementary to A.
S 1 ′ other sequence complementary to A.
S 2 promoter or first replication initiation sequence (origin of replication)
S 2 ′ promoter or second replication initiation sequence (origin of replication)
Ad 1 adapter 1 , complementary to the target nucleic acid
Ad 2 adapter 2 , complementary to the target nucleic acid

The invention is illustrated by the following examples explains:

example 1 Amplification of the tarqet DNA using a detectable labeled deoxvribonucleoside triphosphate (step a)

Polymerase chain reaction is carried out with cloned HBV-specific sequences. The primers used bind to positions 1937-1960 and 2434-2460 in the HBV genome, ie a sequence of 523 nucleotides is amplified (R. Sumazaki et al., 1989, J. Med. Virol, 27, 304-308). The concentration of the DNA to be amplified in the plasmid corresponds to 100 ag to 100 pg in the dilution series used here. 30 PCR cycles (2 ′ 92 ° C; 2 ′ 50 ° C; 2 ′ 70 ° C) are performed in 100 µl with primers, 200 nM each; KCl, 50 mM; Tris HCl pH 8.5, 10 mM; MgCl ₂ , 1.5 mM; Gelatin 100 µg / ml; dATP, 200 µm; dCTP, 200 µm, dCTP, 200 µm; dTTP, 150 µM; Digoxigenin-11-2'-deoxy-uridine-5'-dUTP (DIG- [11] -dUTP, Boehringer Mannheim), 50 µm; 2.5 U Thermus acuaticus (day) DNA polymerase performed.

Non-thermal denaturation (step e))

20 µl of this mixture after PCR and 40 ng of biotin-labeled Sample DNA (randomly primed, cloned HBV DNA between Positions 27 and 2604, double-stranded) are in one Total volume of 44 µl in 0.5 M NaOH 10 'at 37 ° C denatured.

Hybridization with probe C (step b)) and solid phase binding (Step f))

The mixture is then neutralized by adding 156 μl of hybridization solution of pH 5 and pipetted into a streptavidin-coated microtiter plate (hybridization solution = 62.5 mM sodium phosphate, 6.25 × SSC [ 1 × SSC = NaCl, 0.15 M; sodium citrate, 0.015 M ] and formamide 62.5%). The hybridization / wall binding reaction is carried out at 37 ° C for 18 hours with gentle shaking.

Detection of hybrid D (steps c), g) and h))

After removing the liquid and washing 5 times with 0.025% NaCl and 100/0 copper sulfate are 200 mU / ml anti-digoxigenin Horseradish-peroxidase conjugate added and 60 'at 37 ° C in Tris HCl, pH 7.5 / 10 mM; NaCL 0.9%; BSA, 1%; Pluronic T68 0.5% incubated. Wash after 5x (same conditions as above) is with 0.1% 2,2-azino-di- [3-ethylbenzthiazole] - (ABTS) 60 ' Incubated 37 ° C and the absorbance measured at 405 nm.

Fig. 4 shows the extinctions for this reaction and for the control reaction with biomarked sample.

Example 2

The amplification reaction (polymerase chain reaction) is carried out with control serum (HBV-DNA negative), which is enriched with HBV-specific DNA. Due to the location of the selected PCR primers, a 523 bp DNA fragment is formed during the amplification (PCR primer 1: position 1937-1960, PCR primer 2: position 2434-2460 in the HBV genome, for literature see example 1) . A dilution series of the plasmid in a range from 10 pg / ml to 1.25 pg / ml in control serum is prepared. This corresponds, based on the DNA region of the plasmid to be amplified, to amounts in the range from 40 ag to 320 ag DNA, which are used in the amplification reaction. For alkali lysis, 10 µl of the control serum spiked with plasmid DNA are mixed with 50 µl 10 mM Tris-HCl (pH 8.3) and 20 µl 0.5 N NaOH and incubated for 1 h at 37 ° C. After neutralization with 20 µl 0.5 N HCl, 10 µl of the lysis batch are used in the amplification reaction. The reaction takes place in a total volume of 100 μl with 200 nM each of the PCR primers, 200 μM each of dATP, dCTP and dGTP, 175 μM dTTP, 25 μM digoxigenin-11-2′-dUTP (Boehringer Mannheim), 2.5 U Thermus acuaticus DNA polymerase, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 0.01% gelatin. After overlaying the solution with 100 µl mineral oil, the incubation takes place in a thermal cycler (Perkin Elmer): 30 sec 92 ° C, 30 sec 50 ° C, 60 sec 70 ° C, a total of 30 cycles. After the amplification has ended, 40 μl of the mixture are mixed with 10 μl of 0.5 N NaOH and incubated for 10 min at room temperature. The neutralization is carried out by adding 450 μl hybridization solution (50 mM Na phosphate buffer, 0.75 M NaCl, 0.075 M Na citrate, 0.05 H HCl, O, 65% RSA, pH 5.4) to the 220 ng / ml Biotin-labeled oligonucleotide (Bio-Oli 25, biotin-labeled, according to Applied Biosystems, User Bulletin, DNA Synthesizer No. 49, 1988, position 2327-2356 in the HBV genome), which is complementary to a region of the amplified DNA region is. 200 / ul of the hybridization mixture are incubated in a cavity of a microtiter plate coated with streptavidin at 37 ° C. for 3 h. After the solution has been suctioned off, it is then washed 2 × 10 min at 37 ° C. with 0.3 M NaCl, 0.03 M Na citrate, 0.2 96 SDS and 1 × briefly at room temperature with 0.9% NaCl. 200 mU / ml anti-digoxigenin-antibody-horseradish peroxidase conjugate in 100 mM Tris-HCl (pH 7.5), 0.9% NaCl, 1% RSA are added and incubated at 37 ° C. for 30 min. After washing three times with 0.9% NaCl, the substrate solution (1.9 mM ABTS®) is added and the absorbance measured at 405 nm.

The results of the reaction are shown graphically in FIG .

Example 3

HBe-positive human plasma with a virus titer of 1 × 10 ¹⁰ hepatitis B viruses per mol is diluted in normal serum, so that the virus content of the dilutions is between 0.25 × 10 ⁵ / ml to 2 × 10 ⁵ / ml. For the lysis of the viruses, 10 ul of the serum dilution are mixed with 10 ul 0.2 N NaOH and incubated for 1 h at 37 ° C. The subsequent neutralization is carried out by adding 30 μl of neutralization solution (100 mM KCl, 20 mM Tris-HCl (pH 8.3)), 3 mM MgCl ₂ , 0.02% gelatin, mixed with 10 μl of the lysis batch used. The reaction is carried out in a total volume of 100 μl with 200 nM each of the two PCR primers, 200 μm each of dATP, dCTP and dGTP, 175 μM dTTP, 25 digoxigenin-11-2′-dUTP (Boehringer Mannheim), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 0.01% gelatin, 2.5 U Thermus acuaticus DNA polymerase. The reaction mixture is overlaid with 100 μl of mineral oil and incubated in a thermal cycler (Perkin Elmer) for 30 cycles: 30 sec at 92 ° C., 30 sec at 50 ° C. and 1 min at 70 ° C.

A 523 bp DNA fragment is generated by the amplification step (HBV-PCR primer 1: position 1937-1960, HBV-PCR primer 2: position 2434-2460 in the HBV genome). The specific detection of the PCR products is carried out in a two-component test system on the solid phase. First 20 µl of the amplification mixture are mixed with 20 µl TE buffer (10 mM Tris-HCl (pH 7.5) 1 mM EDTA) and 10 µl 0.5 N NaOH and incubated for 10 min at room temperature. The neutralization is carried out by adding 450 μl acidified hybridization solution ( 50 mM Na phosphate buffer, 0.05 N HCl, 0.75 M NaCl, 0.075 M Na citrate, 0.05% RSA, pH 5.4), the 100 ng / ml of a biotin-labeled oligonucleotide is added which corresponds to a region of the amplified DNA region (HBV-biotin oligonucleotide, 30mer, 2-fold biotin-labeled, position 2327-2356 in the HBV genome, Bio-Oli 25, produced by solid-phase synthesis , biotin-labeled as described in Example 2). 200 μl of this hybridization mixture are incubated in a cavity of a microtiter plate coated with streptavidin for 3 hours at 37 ° C. with shaking. After the hybridization and wall binding reaction, the solution is suctioned off and 2 × 10 min with 0.3 M NaCl, 0.03 M Na citrate, 0.2% SDS at 37 ° C. and 1 × briefly with 0.9% NaCl Washed at room temperature. After adding 200 mU / ml anti-digoxigenin-antibody-horseradish peroxidase conjugate in 100 mM Tris-HCl (pH 7.5), 0.9% NaCl, 1% RSA, the mixture is incubated at 37 ° C. with shaking for 30 min. Unbound conjugate is removed by washing 3 times with 0.9% NaCl at room temperature. After 30 minutes of incubation with 1.9 mM ABTS® (2,2'-azino-di- [3-ethyl-benzothiazoline-sulfonic acid (6)] - diammonium salt) at 37 ° C, the absorbance at 405 nm is determined using an ELISA reader measured. The results are shown in Fig. 6.

Claims (24)

1. A method for the specific detection of a nucleic acid in a sample, which comprises the following steps:

• a) reaction of the sample with one or more labeled mononucleoside triphosphates and one or more enzymes which catalyze the production of a labeled nucleic acid B which contains this nucleotide,
• b) reaction of the sample with a nucleic acid probe C, which is sufficiently complementary to nucleic acid B, and
• c) detection of the nucleic acid hybrid D formed from labeled nucleic acid B and nucleic acid probe C,

characterized in that

• d) the nucleic acid probe contains at least one immobilizable group,
• e) the reaction mixture is subjected to a non-thermal denaturation after step a),
• f) the nucleic acid hybrid D is brought into contact with a solid phase which can specifically bind the immobilizable nucleic acid probe C,
• g) the liquid is separated from the solid phase and
• h) the detectable group bound to the solid phase is detected.

2. The method according to claim 1, characterized in that Reaction a) in the presence of a specific starter expires. 3. The method according to claim 1 or 2, characterized in that that the enzyme or the enzyme complex the polymerization of Nucleoside triphosphates to a nucleic acid A im essential complementary nucleic acid B catalyzed. 4. The method according to claim 3, characterized in that the nucleoside triphosphates ribonucleoside triphosphates include. 5. The method according to claim 2, characterized in that a primer P 1 is used in reaction a) as a specific starter, which is essentially complementary to part of nucleic acid A and from the enzyme with incorporation of the mononucleoside triphosphate to one of nucleic acid A. essentially complementary nucleic acid B is extended. 6. The method according to claim 5, characterized in that a primer P 2 is also used which is substantially complementary to a part of the nucleic acid B. 7. The method according to any one of claims 3 or 5, characterized characterized in that the nucleoside triphosphates Deoxyribonucleoside triphosphates include.   8. The method according to any one of claims 1 to 3, characterized characterized in that the nucleic acid to be detected is single-stranded or is made, then the sample at least one adapter per single strand to be verified is added, which one to a part of the nucleic acid to be detected essentially complementary Nucleotide sequence and a replication initiation region contains, the adapter by one to the nucleic acid complementary sequence is extended, and so formed nucleic acid as a template nucleic acid in Reaction a) is used. 9. The method according to any one of claims 1 to 3, characterized characterized in that the nucleic acid to be detected is single-stranded or is made, then the sample at least one primer per single strand to be detected is added, which is a part of the nucleic acid to be detected essentially complementary Nucleotide sequence and a transcription initiation sequence contains, the primer to one to be detected Nucleic acid complementary nucleotide sequence extended and the nucleic acid thus formed as a template nucleic acid in Reaction a) is used. 10. The method according to any one of the preceding claims, characterized characterized in that the one formed in step a) Nucleic acid B as template nucleic acid again in Reaction a) is used. 11. The method according to any one of the preceding claims, characterized characterized in that reaction a) several times in succession is carried out, each of the products of the reaction again serve as starting materials for the new reaction.   12. The method according to claim 10 or 11, characterized characterized in that in reaction a) a nucleic acid hybrid is formed from nucleic acid A and nucleic acid B. 13. The method according to any one of the preceding claims, characterized in that the denaturation at alkaline pH takes place. 14. The method according to any one of the preceding claims, characterized characterized in that the denaturation by addition chaotropic salts is made. 15. The method according to claim 13 or 14, characterized characterized in that the denaturation in one Temperature range between 17 ° C and 50 ° C takes place. 16. The method according to any one of claims 13, 14 or 15, characterized in that the entire detection procedure in a temperature range between 17 ° C and 50 ° C takes place. 17. The method according to claim 16, characterized in that the entire detection procedure with a single Temperature takes place. 18. The method according to claim 11, characterized in that the nucleic acid probe in a solution with a pH between 3.0 and 6.5 is added. 19. The method according to claim 18, characterized in that the solution also all for carrying out the Contains the necessary reagents.   20. The method according to claim 18 or 19, characterized in that the nucleic acid probe C is a single-stranded nucleic acid C 1 , the opposite strand of which is not present in the solution. 21. The method according to claim 13, characterized in that the nucleic acid probe C in a solution with a pH between 10 and 14 is added and then with the Hybridization solution in the mixture a pH between 5.0 and 8.5 is set. 22. The method according to claim 13, characterized in that first a solution of the probe is combined with the sample, then a pH between 10 and 14 is set and then a pH between 5.0 and 8.5 in the mixture is set. 23. The method according to claim 6, characterized in that the nucleic acids formed by extension of the primers P 1 and P 2 are reacted again with P 1 and P 2 after strand separation, the strands newly formed with the one primer each as a template for the extension serve the other primer.