JP2005137359A - Method for suppressing nonspecific hybrid formation in method for extending primer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To suppress nonspecific extension reaction of a primer in a method for extending the primer. <P>SOLUTION: The method of the invention is the primer extension reaction by forming a nucleic acid chain complementary to a template chain of the nucleic acid by the primer. The primer is reacted with the template chain in the presence of the primer connected with the nucleic acid complementary to 3' end of the primer and having affinity to the primer equal to or less than the affinity of the primer to the template chain of the nucleic acid or its derivative. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Background of the Invention

The present invention relates to a method for suppressing nonspecific extension of primers in a primer extension method.

Background Art A primer extension reaction that forms a nucleic acid complementary to a template of a nucleic acid not only plays an important role in vivo, but is also utilized as an indispensable technique in genetic engineering. That is, a template and a primer complementary thereto form a double strand, and a polymerase adds a mononucleotide to the 3 ′ end of the primer in a complementary manner to the template. Vary et al. Have proposed a nucleic acid detection method using the above template-dependent primer extension reaction (US Pat. No. 4,851,331).

  Various gene amplification methods using this primer extension method have been developed (for example, PCR method, SDA method, RCR method, 3SR method, NASB method: Keller, GH et al., DNA PROBES, p255-297, Stockton press (1993 ), Persing, DH et al., Diagnastic Molecular Microbilogy, p51-87, American Society for Microbiology (1993)), which has made tremendous progress in the field of genetic engineering.

  However, in gene amplification reactions using these primer extension methods, in order to amplify a specific gene about 1 million times, a reagent (polymerase) involved in the reaction for the gene to be amplified in the initial process of the reaction. , Primers, unit nucleic acids, etc.) in large excess. Therefore, non-specific hybridization is very likely (Chou et al., Nucleic Acids Res., 20,1717 (1992)). Even in the PCR method that seems to be the best in specificity, amplification is performed with high specificity when the amount of template nucleic acid is very small and when a large amount of sample-derived impurities are mixed. That was not easy. Furthermore, it was found that the PCR method had an unexpected primer / dimer problem (Li et al., Proc. Natl. Acad. Sci. USA. 87, 4580-4584 (1990)).

  In order to solve these problems, the Hot-Start method (Chou et al., Nucleic Acids Res, 20, 1717-1723 (1992)) and the method using an antibody against a polymerase (Kellogg et al., Bio Techniques 16,1134) -1137 (1994), Sharkey et al., BIO / TECHNOLOGY 12,506-507 (1994)), etc., but both methods sufficiently suppress non-specific hybridization and extension after initiation of the PCR reaction. And cannot be applied to amplification methods other than the PCR method. As a reliable method to improve specificity, there is a nested PCR method (Pierre et al., J. Clin. Microbiol. 29, 712-717 (1991)) that uses two sets of primers. It is not considered.

Summary of the Invention

  The object of the present invention is to provide a method for suppressing nonspecific hybridization in the primer extension method.

  Another object of the present invention is to provide a reagent that suppresses non-specific hybridization of the primer to the template strand of the nucleic acid in the primer extension method.

Primer extension reaction to form a nucleic acid strand complementary to the template strand of the nucleic acid by the primer according to the present invention is as follows:
Primer and template in the presence of a nucleic acid (primer complementary nucleic acid) or derivative thereof that is complementary to the primer and whose affinity for the primer is equal to or less than the affinity of the primer for the template strand of the nucleic acid It is characterized by performing a reaction with a chain.

  The reagent for suppressing nonspecific hybridization to the template strand of the nucleic acid of the primer according to the present invention is complementary to the specific primer, and its affinity for the primer is the affinity of the primer for the template strand of the nucleic acid. A nucleic acid or a derivative thereof equivalent to or lower than

  According to the present invention, nonspecific hybridization is suppressed in the primer extension reaction. This seems to be due to the non-specific hybridization of the primer nucleic acid to the template strand due to the presence of the nucleic acid complementary to the primer.

DETAILED DESCRIPTION OF THE INVENTION

  As used herein, “nucleic acid” refers to a nucleic acid comprising ribonucleotides and / or deoxyribonucleotides, and includes DNA, RNA, oligonucleotides mixed with ribonucleotides and deoxyribonucleotides, and the like.

  In the present specification, “nucleic acid derivative” refers to an atom (for example, a hydrogen atom, an oxygen atom) or a functional group (for example, a hydroxyl group or an amino group) such as a base portion, a ribose portion, or a phosphodiester bond portion of a nucleic acid. Is protected with another atom (for example, a hydrogen atom, sulfur atom), a functional group (for example, an amino group), or an alkyl group having 1 to 6 carbon atoms or a protecting group (for example, a methyl group or an acyl group) Or those in which these parts are replaced with non-naturally occurring components (eg, peptide bonds).

  As such a derivative, for example, a peptide nucleic acid (PNA) in which base portions are linked by a peptide bond (Nielsen et al., J. Amer. Chem. Soc., 114,9677-9678 (1992)) exists in nature. Rare nucleic acids (Nucleic Acids Reseach, Vol.22, No12, 2183 (1994)), nucleic acids in which the hydrogen atom of the base amino group is replaced with an alkyl group having 1 to 6 carbon atoms, and the configuration of the hydroxyl group of the ribose moiety is changed. And nucleic acids in which the oxygen atom of the phosphodiester bond portion is substituted with a sulfur atom.

  Further, in the present specification, the “primer” refers to a nucleic acid molecule and a derivative thereof required for a nucleic acid biosynthesis initiation reaction. Therefore, not only the DNA and RNA but also derivatives thereof can be used as the primer.

  The method according to the present invention is basically a primer extension reaction in which a primer forms a nucleic acid strand that is complementary to the template strand of the nucleic acid, and the reaction between this primer and the template strand is complementary to the primer and It is performed in the presence of a nucleic acid (primer complementary nucleic acid) or a derivative thereof whose affinity is equal to or less than the affinity of the primer for the template strand of the nucleic acid. Hereinafter, “primer-complementary nucleic acid” is used in a sense including its derivatives.

  Although not limited by the following theory, the reason why the formation of a non-specific hybrid is suppressed in the present invention is considered as follows. First, the non-specific hybrid is considered to be a phenomenon in which the primer hybridizes to a portion other than the target region where the primer originally forms a hybrid, and a sequence other than the target sequence is formed from that portion. Here, if a primer-complementary nucleic acid that is complementary to the primer and has an affinity equal to or less than the affinity of the primer for the template strand is present, the primer-complementary nucleic acid hybridizes to the originally intended region. It is thought that the primer which did not exist is caught and a hybrid is formed. As a result, the opportunity for the primer to hybridize to a portion other than the target region is reduced, and this is considered to suppress the formation of non-specific hybrids. Since the affinity of the primer-complementary nucleic acid for the primer is equal to or lower than the affinity of the template strand for the primer, this primer-complementary nucleic acid does not adversely affect the formation of a hybrid for the target region of the primer.

  Here, the “affinity” can be expressed using, for example, heat-dependent stability represented by the melting temperature Tm (Higgins et al., Nucleic acid hybridization p80, IRL PRESS (1985)). That is, when Tm is large, the affinity is high, and when Tm is low, the affinity is low.

  As a method for reducing the affinity between the primer-complementary nucleic acid and the primer, there is a method of shortening the chain length of the primer-complementary nucleic acid as compared with the primer. Since the region closer to the 3 'end of the primer is considered to be more important for non-specific hybridization with the template, it is preferable to shorten the chain length from the 3' end side of the primer-complementary nucleic acid. The chain length of the primer-complementary nucleic acid can be appropriately determined in consideration of affinity. For example, when the primer chain length is 1, 1 to 0.4 is preferable, and 0.9 to 0.6 is more preferable. It is.

  The decrease in affinity can also be achieved by introducing nucleotides that are not complementary to the primer into the primer-complementary nucleic acid. In addition, by introducing a base that is complementary to the base of the primer but forms a complementary strand by weaker hydrogen bonds, such as inosine (Martin et al., Nucleic Acids Res. 13, 8927-8938 (1985)). Can also lower the affinity.

  The primer-complementary nucleic acid according to the present invention is preferably inactivated with respect to primer extension activity because it can itself be a primer. Examples of the inactivation method include deoxylation at the 3 ′ end (for example, the 3 ′ terminal nucleotide is changed to 2 ′, 3′-dideoxyribonucleotide) or a protecting group (for example, a methyl group or an acyl group). Protection and the like.

  According to a preferred embodiment of the present invention, the primer and the primer-complementary nucleic acid may be combined to form a single molecule. If both are on the same molecule, that is, if the primer-complementary nucleic acid is physically close, if the primer does not hybridize to the target template strand, the primer-complementary nucleic acid part can be easily To form an intramolecular bond. Thereby, nonspecific hybridization can be efficiently suppressed.

  Accordingly, “one molecule” refers to a state in which a primer-complementary nucleic acid is directly or indirectly bound to a primer in a manner other than hybridization (eg, covalent bond).

  As a method for forming a single molecule, a method that does not lower the hybridization ability of the primer and the primer-complementary nucleic acid is preferable. Examples of the binding position that does not decrease the hybridization ability include a 5′-terminal hydroxyl group, a base part, a phosphodiester part, etc. for the primer, and a 5′- or 3′-terminal hydroxyl group, a base part, etc. for the primer-complementary nucleic acid. Examples thereof include a phosphoric acid diester moiety.

  Specific examples of a method for forming a single molecule include a method in which an oligonucleotide is modified (Ecrstein, Oligonucleotides and Analogues, IRL PRESS (1991)), a linker is introduced, and both are crosslinked with a commercially available crosslinking agent.

  The length of the linker may be any length that does not interfere with the hybridization between the primer and the primer-complementary nucleic acid, and varies depending on the length of the primer and the primer-complementary nucleic acid or the cross-linking position, for example, 3 to 50 atoms (preferably 5 What is necessary is just about 30 atoms apart. The constituent elements of the linker are not particularly limited as long as they do not affect the hybridization between the primer and the primer-complementary nucleic acid or the primer extension reaction.

  Examples of the linker include a saturated or unsaturated hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, and a heterocyclic group. As a saturated hydrocarbon group, a C3-C50 alkyl group is mentioned, A C5-C30 alkyl group is preferable, More preferably, it is a C5-C10 alkyl group.

  The linker may contain an ester bond, an ether bond, a peptide bond, an oxygen atom, a sulfur atom, or a nitrogen atom alone or in combination, and the one or more hydrogen atoms may be a carboxyl group, an amino group, or an amino group. A group, an alkoxy group, an acyl group, an alkoxycarbonyl group, an acyloxy group, a hydroxyl group, or a halogen atom. The linker may also be linear or contain a branched chain. The linker is preferably hydrophilic. For example, the linker may contain an ether bond or a peptide bond.

  As a specific crosslinking method, a linker having an amino group and a thio group is introduced into both nucleic acids, and a bifunctional crosslinking agent (for example, N- (6-maleimido-caproyloxy) succinimide (EMCS), N-succinimidyl) is used. The method of bridge | crosslinking using -3- (2-pyridyl dithio) propionate (SPDP)) is mentioned. In addition, using a reagent that can introduce a spacer between nucleotides (Nelson et al., Nucleic Asids Res 20,6253-6259 (1992)), a primer and a primer-complementary nucleic acid are continuously synthesized using a DNA synthesizer. Is mentioned.

  In the present invention, the “primer extension reaction” refers to a reaction in which a nucleic acid complementary to a template strand of a nucleic acid is added to the 3 ′ end of a primer by a polymerase. As a specific reaction using the primer extension method, Polymerase chain reaction method (PCR method), Self-sustained Sequence Replication method (3SR method), Nucleic Acid based amplification method (NASBA method), Repair chain Reaction method (RCR method), Strand Displacement Amplification method (SDA method), Polymerase / Examples thereof include gene amplification methods such as Ligase Chain Reaction method (P / LCR method) and Sanger method.

  The primer extension reaction can be performed in a reaction solution containing a nucleic acid template strand, primer, polymerase, mononucleotide and the like. First, the reaction solution is placed under hybridization forming conditions (for example, the melting temperature or lower), and then the primer is extended by working a polymerase.

  The amount of the primer-complementary nucleic acid in the reaction solution is appropriately determined in consideration of the specific binding of the primer to the template and the suppression of non-specific binding. For example, the amount is preferably 1 mole or more with respect to 1 mole of the primer. Preferably it is 2-10 mol.

  The reaction solution may contain a nucleic acid template strand, polymerase, mononucleotide and the like. The template strand of the nucleic acid may be DNA or RNA, or a derivative thereof. Examples of the polymerase that can be used include, but are not limited to, DNA polymerase, RNA-dependent DNA polymerase (reverse transcriptase), RNA polymerase, and the like. Examples of the mononucleotide include ribonucleotides or deoxyribonucleotides or derivatives thereof (for example, those in which the 3 'hydroxyl group is deoxylated), or a mixture thereof.

  In the method for suppressing nonspecific hybridization, the primer-complementary nucleic acid may be preliminarily present in the reaction solution or may be added immediately before the primer extension reaction.

  Furthermore, according to another aspect of the present invention, a nucleic acid or a derivative thereof that is complementary to a specific primer and whose affinity for the primer is equal to or less than the affinity of the primer for the template strand of the nucleic acid. A reagent for suppressing nonspecific hybridization to a template strand of a nucleic acid of a primer in the primer extension method is provided.

  The “specific primer” is relatively determined in relation to the nucleic acid serving as a template in the reagent, and accordingly, a nucleic acid complementary to the primer is also determined correspondingly. For example, when the template nucleic acid is the protein A gene of Staphylococcus aureus, the specific primer is a nucleic acid having a sequence complementary to the gene, such as SPA1 and SPA2 described in Example 1. Applicable.

  The amount of the nucleic acid complementary to the above-mentioned primer in the reagent for inhibiting nonspecific hybridization and what may be contained in the reagent can be the same as described above.

  EXAMPLES Next, although an Example is given and this invention is demonstrated, this invention is not limited to these Examples.

The synthesis of oligodeoxyribonucleotides in the present invention was performed using an automatic DNA synthesizer model 381A (Applied Biosystems) based on the phosphoramidite method (Tetrahedron Lett., 22, 1859 (1981)) of Caruthers et al. . In addition, for oligonucleotides having a labeled product introduced at the 5 ′ end, first, each 5 ′ terminal amino group is introduced into the oligonucleotide, and then labeled using an appropriate labeling reagent (eg, a bithion group, a dinitrophenol group, etc.). It was prepared by. Furthermore, an oligonucleotide having an amino group introduced at the 3 ′ end was prepared according to the method of Nelson et al. (Nelson et al., Nucleic Acids Res., 17 , 7187-7194 (1989)). The PCR reaction was performed with reference to the method described in PCR Protocols (Innis, MASAcademic Press (1990)).

  Amplification in the PCR reaction was performed by the ED-PCR method (Japanese Patent Laid-Open No. 1-252300).

Example 1 Detection of a specific gene by PCR As a detection of a specific gene, a protein A gene peculiar to S. aureus was detected (Shuttleworth, HL et al., Gene 58, 283-295 (1987)). Using a pair of primers shown below, a part of the gene encoding 224 base pair protein A was specifically amplified by PCR and confirmed by agarose electrophoresis.
SPA1: 5'-BIO-TACATGTCGTTAAACCTGGG-3 '(SEQ ID NO: 1)
SPA2: 5'-DNP-TACAGTTGTACCGATGAATGG-3 '(SEQ ID NO: 2)

  Here, biotin group (BIO) was introduced at the 5 'end for SPA1, and dinitrophenol group (DNP) was introduced at the 5' end for SPA2.

  The detection method of the PCR product by the ED-PCR method using the above primers was as follows.

First, a PCR reaction solution was prepared as follows.
10 × Amplitaq (trade name) reaction solution 5 μl
20 mM dNTP 0.5 μl
SPA1 50 ng / μl 1 μl
SPA2 50 ng / μl 1 μl
Amplitaq (trade name) polymerase (1.25 U / μl) 1 μl
41.5 μl of H ₂ O

PCR reaction was carried out on those to which S. aureus DNA (400 pg / μl; 1 μl) was added and nothing was added thereto. SPA1 and SPA2 were used as primers. The temperature conditions were 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 60 seconds for one cycle. 10 μl of the reaction solution was reacted with the alkaline phosphatase labeled antibody in a streptavidin plate. After 30 minutes, the reaction solution was removed and the plate was washed 3 times with a washing solution. Finally, a buffer solution and a chromogenic substrate (1M diethanolamine (pH 9.8), 0.5 mM MgCl ₂ , 4 mg / ml p-nitrophenyl phosphate) were added to perform a color reaction, and the absorbance was measured using a plate reader. .

  As a result, the color development after 10 minutes was 0.01 when nothing was added as a template, and 1.80 when 400 pg of S. aureus DNA was added as a template. On the other hand, when the PCR reaction was performed at 40 cycles, the color development after 10 minutes was 1.00 when nothing was added as a template, and when 400 pg of S. aureus DNA was added as a template. It was 1.93.

  Thus, when the number of PCR reaction cycles was increased from 35 to 40, it became difficult to determine whether S. aureus was positive or negative.

Example 2 Suppression of non-specific extension reaction in PCR method (1)
Oligonucleotides complementary to the primers SPA1 and SPA2 were synthesized, and an attempt was made to suppress nonspecific extension reaction in the PCR method.
SPA1-C1: 5′-CACCAGGTTTAC-3′NH ₂ (SEQ ID NO: 3)
SPA2-C1: 5'-CCATTCATCGGTA- 3'NH 2 ( SEQ ID NO: 4)

SPA1-C1 is complementary to 13 bases from the 3 ′ end of primer SPA1, and SPA2-C1 is complementary to 13 bases from the 3 ′ end of primer SPA2. An amino group (NH ₂ ) was introduced at the 3 ′ end of the oligonucleotide complementary to each primer.

  The PCR reaction solution shown below was prepared, and PCR reaction was performed using SPA1 and SPA2 as primers. The PCR reaction was performed at 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 60 seconds under the conditions of repeating 40 cycles.

Reaction solution 1
10 × Amplitaq (trade name) reaction solution 5 μl
20 mM dNTP 0.5 μl
SPA1 50 ng / μl 1 μl
SPA2 50 ng / μl 1 μl
Amplitaq (trade name) DNA polymerase (1.25 U / μl)
1 μl
SPA1-C1 500 ng / μl 5 μl
SPA2-C1 500 ng / μl 5 μl
H ₂ O 31.5 μl

Reaction solution 2
10 × Amplitaq (trade name) reaction solution 5 μl
20 mM dNTP 0.5 μl
SPA1 50 ng / μl 1 μl
SPA2 50 ng / μl 1 μl
────────────────────────────────────
Amplitaq (trade name) DNA polymerase 1.25 U / μl 1 μl
SPA1-C1 500 ng / μl 10 μl
SPA2-C1 500 ng / μl 10 μl
────────────────────────────────────
H ₂ O 21.5 μl

Reaction solution 3
10 × Amplitaq (trade name) reaction solution 5 μl
20 mM dNTP 0.5 μl
SPA1 50 ng / μl 1 μl
SPA2 50 ng / μl 1 μl
Amplitaq (trade name) DNA polymerase 1 μl
41.5 μl of H ₂ O

  With respect to each reaction solution, PCR reaction was performed when S. aureus DNA (400 pg / μl: 1 μl) was added and when it was not added, and the absorbance was measured in the same manner as in Example 1.

As a result, color development after 12 minutes was as follows.
Reaction liquid 1 Reaction liquid 2 Reaction liquid 3
When nothing is added as a mold 0.05 0.04 1.57
When S. aureus DNA is added as a template 2.12 1.22 2.47

Example 3 Suppression of non-specific extension reaction in PCR method (2)
SPA1-C2 and SPA2-C2 were used as oligonucleotides complementary to the primers SPA1 and SPA2, and an attempt was made to suppress nonspecific extension reaction in the PCR method.
SPA1-C2: 5′-CACCAGGTTAACGACAT3′-NH ₂ (SEQ ID NO: 5)
SPA2-C2: 5'-CCATTCATCGGTACAACT3'- NH 2 ( SEQ ID NO: 6)

  SPA1-C2 is complementary to 18 bases from the 3 'end of primer SPA1, and SPA2-C2 is complementary to 18 bases from the 3' end of primer SPA2.

  A reaction solution shown below was prepared, and a PCR reaction was performed using SPA1 and SPA2 as primers. The PCR reaction was performed at 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 60 seconds under the conditions of repeating 40 cycles.

Reaction solution 4
10 × Amplitaq (trade name) reaction solution 5 μl
20 mM dNTP 0.5 μl
SPA1 50 ng / μl 1 μl
SPA2 50 ng / μl 1 μl
Amplitaq (trade name) DNA polymerase (1.25 U / μl)
1 μl
41.5 μl of H ₂ O

Reaction solution 5
────────────────────────────────────
10 × Amplitaq (trade name) reaction solution 5 μl
20 mM dNTP 0.5 μl
SPA1 50 ng / μl 1 μl
────────────────────────────────────
SPA2 50 ng / μl 1 μl
Amplitaq (trade name) DNA polymerase (1.25 U / μl)
1 μl
SPA1-C2 500 ng / μl 1 μl
SPA2-C2 500 ng / μl 1 μl
39.5 μl of H ₂ O

  Each reaction solution was subjected to a PCR reaction in the same manner as in Example 2, and the absorbance was measured. In addition, the same experiment was performed on human DNA (700 ng / reaction) added as a template.

As a result, color development after 10 minutes was as follows.
Reaction liquid 4 Reaction liquid 5
When nothing is added as a mold 0.35 0.00
When 700 ng of human DNA is added as a template 0.09 0.00
When 400 ng of S. aureus DNA is added as a template 2.21 1.16

Example 4 Suppression of non-specific extension reaction in PCR method (3)
(1) Synthesis of primer combined with primer-complementary nucleic acid A primer combined with a primer-complementary nucleic acid represented by the following formula was synthesized.
SPA1C:
5'Bio-CACCAGGTTTAC- (linker) -TACATGTCGTTAAACCTGGTG (SEQ ID NO: 7)
SPA2C:
5'DNP-CCATCATCATGGTA- (linker) -TACAGTTGTTACCGATGAATGG (SEQ ID NO: 8)

First, in synthesizing SPA1, first, a primer portion was synthesized. In order to introduce a thiol after adding the above sequence sequentially from the 3 ′ end and adding a base at the 5 ′ end of the primer, the primer portion is a C6-thiol modifier (trade name) (in accordance with general oligonucleotide synthesis). Clontech) was added. After completion of the synthesis, elimination from the carrier and removal of the protecting group were carried out as usual, and crude purification was carried out by gel filtration (Sephadex G-50, 50 mM TEAB buffer pH 7.2). Crude product 10 A260 units were evaporated to dryness and dissolved in 0.1 M TEAA (triethylammonium acetate), pH 7.5 (100 μl). To this, 1.0M AgNO ₃ (15 μl) was added and reacted at room temperature for 30 minutes. Next, 1.0 M DTT (dithiothreitol) (10 μl) was added and reacted at room temperature for another 15 minutes. After the reaction, the precipitate was removed by centrifugation and purified by gel filtration (Sephadex G-50, 40 mM phosphate buffer, pH 6.0), and the eluate was used as it was in the next reaction.

On the other hand, the portion complementary to the primer was synthesized as follows. In order to introduce an amino group at the 3 ′ end, a base sequence of a portion complementary to the primer was sequentially added using a carrier of Nelson's method (Nelson, Nucleic Acids Res., 17, 7187-7194) as a raw material. After the last base was added, biotin ON phosphoramidite (Clontech) was added to introduce biotin at the 5 'end. After completion of the synthesis, elimination from the carrier and removal of the protecting group were carried out as usual, and crude purification was carried out by gel filtration (Sephadex G-50, 50 mM TEAB buffer pH 7.2). Crude product 10 A260 units were evaporated to dryness and dissolved in H ₂ O (40 μl). 1M NaHCO ₃ (10 μl) was added, and a DMF solution (20 mg / ml, 50 μl) of EMCS (DOJINDO) was further added, followed by reaction at room temperature for 5 hours. After the reaction, the reagent was removed by gel filtration (Sephadex G-50, 50 mM TEAB buffer, pH 7.2).

  The two kinds of oligonucleotides were bound as follows. First, biotin at the 5 'end and an oligonucleotide having an amino group introduced at the 3' end were evaporated to dryness, and the total amount of the phosphate buffer solution of the oligonucleotide introduced with a thiol group at the 5 'end was added for 16 hours at room temperature. Reacted. After the reaction, the buffer was exchanged by gel filtration (Sephadex G-50, 50 mM TEAB buffer pH 7.2) and evaporated to dryness. The resulting mixture was purified by 20% polyacrylamide electrophoresis containing 8.3 M urea. Oligonucleotides were collected from the band with the lower mobility to obtain SPA1C primer bound with primer-complementary nucleic acid.

  The SPA2C primer was synthesized in the same manner, but DNP-ON phosphoramidite (Clontech) was used to synthesize a portion complementary to the primer in order to introduce DNP at the 5 'end.

(2) Suppression of non-specific amplification reaction by primers SPA1C and SPA2C The following reaction solutions were prepared, and the effect of suppressing the non-characteristic reaction of the primer bound with a nucleic acid complementary to the primer was examined.
10 × Amplitaq (trade name) reaction solution 5 μl
20 mM dNTP 0.5 μl
SPA1C 100 ng / μl 1 μl
SPA2C 100 ng / μl 1 μl
Amplitaq (trade name) DNA polymerase (1.25 U / μl) 1 μl
41.5 μl of H ₂ O

The absorbance was measured by carrying out a PCR reaction in the same manner as in Example 2 with respect to the above-mentioned reaction solution containing S. aureus DNA added as a template, nothing added as a template, and DNA added as a template. As a result, the issue after 10 minutes was as follows.
When nothing is added as a mold 0.00
When human DNA (700 ng) is added as a template 0.00
When S. aureus DNA (400 ng) is added as a template 0.53

  As described above, nonspecific amplification reaction could be suppressed, but specific amplification was also suppressed.

(3) As a result of primer binding nucleic acid complementary to primer and shortening its chain length Primer binding nucleic acid complementary to primer shown below and having its chain length shortened ( Synthesis was performed in the same manner as in 1), and the results were examined.
SPA1CS:
5'Bio-CACCAGGT- (linker) -TACATGTCGTTAAACCTGGGTG (SEQ ID NO: 9)
SPA2CS:
5'DNP-CCATCATCAT- (linker) -TACAGTTGTACCGATGAATGG (SEQ ID NO: 10)

A PCR reaction was performed under the same conditions as in (2), and the absorbance was measured.
When nothing is added as a mold 0.00
When human DNA (700 ng) is added as a template 0.00
When S. aureus DNA (400 ng) is added as a template 2.15

  As described above, by shortening the chain length of the nucleic acid complementary to the primer, it was possible to efficiently carry out the specific reaction while suppressing the nonspecific amplification reaction.

The present invention is as follows.
(1) A primer extension reaction in which a primer forms a nucleic acid strand complementary to a template strand of nucleic acid,
Primer and template in the presence of a nucleic acid (primer complementary nucleic acid) or derivative thereof that is complementary to the primer and whose affinity for the primer is equal to or less than the affinity of the primer for the template strand of the nucleic acid Performing the reaction with a chain.
(2) The method according to (1), wherein the primer, the primer-complementary nucleic acid, and the template strand are each independently DNA or RNA.
(3) The method according to (1) or (2), wherein the primer-complementary nucleic acid or derivative thereof has a shorter chain length than the primer.
(4) The method according to any one of (1) to (3), wherein the primer-complementary nucleic acid or derivative thereof includes a nucleotide that is not complementary to the primer in a part thereof.
(5) The method according to any one of (1) to (4), wherein the primer-complementary nucleic acid or derivative thereof comprises a nucleotide that forms a weak hydrogen bond with the primer.
(6) The method according to (5), wherein the nucleotide that weakly bonds with a primer is inosine.
(7) The method according to any one of (1) to (6), wherein the primer and the primer-complementary nucleic acid or derivative thereof are combined to form one molecule.
(8) The method according to (7), wherein the primer and the primer-complementary nucleic acid or derivative thereof are linked by a linker.
(9) The method according to (8), wherein the linker is selected from the group consisting of a saturated or unsaturated hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, and a heterocyclic group. .
(10) The method according to (9), wherein the linker is a saturated hydrocarbon group.
(11) The method according to (10), wherein the linker is an alkyl group having 5 to 30 carbon atoms.
(12) The derivative of the primer-complementary nucleic acid is any one of (1) to (11), wherein the hydroxyl group at the 3 ′ end is substituted with a hydrogen atom or a functional group, or is protected with a protecting group The method according to one item.
(13) The method according to any one of (1) to (12), wherein the derivative of the nucleic acid complementary to the primer is a peptide nucleic acid (PNA).
(14) The primer extension method is selected from the group consisting of polymerase chain reaction method (PCR method), 3SR method, NASBA method, RCR method, SDA method, P / LCR method, and Sanger method, (1 ) To (13).
(15) In a primer extension method comprising a nucleic acid or a derivative thereof which is complementary to a specific primer and whose affinity for the primer is equal to or less than the affinity of the primer for the template strand of the nucleic acid A reagent for suppressing nonspecific hybridization to a template strand of a nucleic acid of a primer.

Claims (9)

A primer extension method in which a primer forms a nucleic acid strand complementary to a template strand of a nucleic acid,
A nucleic acid (primer complementary nucleic acid) or a derivative thereof that is complementary to the 3 ′ end of the primer and whose affinity for the primer is equal to or less than the affinity of the primer for the template strand of the nucleic acid is linked. And reacting the primer with the template strand in the presence of the primer.   The method according to claim 1, wherein the primer, the primer-complementary nucleic acid, and the template strand are each independently DNA or RNA.   The method according to claim 1 or 2, wherein the primer-complementary nucleic acid or derivative thereof has a shorter chain length than the primer.   The method according to any one of claims 1 to 3, wherein the primer-complementary nucleic acid or derivative thereof contains a nucleotide that is not complementary to the primer in part.   The method according to any one of claims 1 to 4, wherein the primer-complementary nucleic acid or derivative thereof comprises a nucleotide that weakly hydrogen-bonds with the primer.   The method according to any one of claims 1 to 5, wherein the primer and the primer-complementary nucleic acid or derivative thereof are linked directly or indirectly.   The method according to any one of claims 1 to 6, wherein the primer and the primer-complementary nucleic acid or derivative thereof are linked by a linker.   The method according to any one of claims 1 to 7, wherein the derivative of the nucleic acid complementary to the primer is a peptide nucleic acid (PNA).   The primer extension method is selected from the group consisting of polymerase chain reaction method (PCR method), 3SR method, NASBA method, RCR method, SDA method, P / LCR method, and Sanger method. The method as described in any one of.