JP6318813B2 - PCR method using MutS mutant - Google Patents

Description

The present invention relates to the field of nucleic acid amplification. More specifically, the present invention relates to a method for suppressing the generation of nonspecific reaction products in nucleic acid amplification.

Synthesis of DNA from a template nucleic acid using a DNA polymerase is used and applied to various methods such as sequencing and nucleic acid amplification in the field of molecular biology. Among them, the nucleic acid amplification method has already been put into practical use not only in the research field, but also in the forensic field such as genetic diagnosis and parentage testing, or in the inspection of microorganisms in food and the environment.

  Numerous nucleic acid amplification methods have been developed so far, such as the polymerase chain reaction (PCR) method, the Loop-Mediated Isotropic Amplification (LAMP) method, the Transcribation Reverse Transcribation Concluded Reaction Reaction (TRC), and the Nucleic Acid Amplification method. Nucleic acid amplification methods such as the (NASBA) method are relatively popular.

Among these, the PCR method used for amplification of a specific sequence of DNA is a technique that is very widespread from research fields to application fields. Currently, the PCR method is being further developed, and there are various technologies such as the Multiplex PCR method that simultaneously amplifies multiple primers, and the real-time PCR method that detects fluorescent amplification products in real time using a fluorescent dye. To do. These techniques have already been put into practical use not only in the research field, but also in the forensic field, food, and environmental microbiological tests.

The gene amplification method by the PCR method is performed by repeating 20 to 50 cycles consisting of denaturation, annealing, and extension in the presence of a target nucleic acid, four types of deoxynucleoside triphosphates, at least a pair of primers and a DNA polymerase. In this method, a region of a target nucleic acid sandwiched between a pair of primers is exponentially amplified.

The PCR method has the feature that the target gene sequence can be amplified by amplifying the region of the target nucleic acid sandwiched between a pair of primers, while the primer has hybridized to the target sequence (primer (Mismatch annealing of the above) also has the disadvantage that amplification of the sequence occurs and non-specific amplification occurs. In addition, when primers are associated with each other during PCR, a primer dimer is generated, which acts as a template DNA to generate a nonspecific amplification product. Therefore, the problem in PCR is to suppress non-specific amplification and amplify only the target product.

As a method for reducing non-specific amplification, Patent Document 1 describes a method for reducing non-specific amplification by adding heat-stable MutS (MutS mismatch binding protein (MutS DNA Mismatch-Binding Protein)) to PCR. . Non-patent document 1 reports that nonspecific amplification during isothermal amplification was reduced using MutS derived from Thermus aquaticus. In these methods, MutS recognizes and binds to mismatched base pairs generated during amplification, thereby inhibiting non-specific amplification by inhibiting the action of DNA polymerase, thereby reducing non-specific amplification in nucleic acid amplification reactions. It is supposed to be possible.

  However, when MutS was added, it was frequently observed that nucleic acid amplification other than non-specific amplification was observed, and further improvement was required.

WO2013 / 175815

Mitani, Y. et al. et al. Nat Methods 4, 257-262 (2007) M.M. H. Lamers et al. The EMBO Journal 22, 746-756 (2003) G. Obmolova et al. Nature 407, 703-710 (2000) Takamatsu, S et al. Nucleic Acids Res 24, 640-647 (1996) M.M. S. Junop et al. Molecular Cell 7, 1-12 (2001) Yokoyama et al. Nature Struct. Biol. 7,943-945 (2000)

An object of the present invention is to reduce the inhibition of amplification due to the addition of MutS in the nucleic acid amplification reaction and to improve the effect of suppressing the nonspecific amplification reaction of MutS.

The present inventors have found a result suggesting that the inhibition occurring in the nucleic acid amplification reaction with the addition of MutS is caused by the inactivation of nucleoside triphosphates by MutS.
As a result of investigating a method for solving this problem, it was found that the use of a MutS mutant with a modified ATPase active site reduces amplification inhibition due to the addition of MutS, thereby completing the present invention.

That is, the present invention has the following configuration.
[1]
A nucleic acid amplification method comprising a MutS mutant having reduced inactivation activity for nucleoside triphosphate in a reaction solution.
[2]
The method according to [1], wherein the MutS mutant is a MutS mutant having a modified ATPase active site.
[3]
The method according to [1] or [2], wherein the MutS mutant is a MutS mutant in which a polar amino acid residue of an ATPase active site is modified to a nonpolar amino acid residue or an amino acid residue having an opposite polarity.
[4]
The MutS variant is a MutS variant having a modification of at least one amino acid among amino acids related to the ATPase active site consisting of amino acids corresponding to the 597th, 670th, and 671th amino acids in the amino acid shown in SEQ ID NO: 1. The method according to any one of [1] to [3].
[5]
The method according to [4], wherein MutS corresponding to K597 is an amino acid substitution of K597M or K597A.
[6]
The method according to [4], wherein the modification corresponding to D670 uses MutS, which is an amino acid substitution of D670A.
[7]
The method according to [4], wherein the modification corresponding to E671 uses MutS, which is an amino acid substitution of E671A, E671M, E671K, or E671N.
[8]
A reaction liquid composition for use in the method according to any one of [1] to [7].
[9]
The reaction liquid composition according to [8], comprising the following (a) to (e):
(A) DNA polymerase (b) a pair of primers in which one primer is complementary to the DNA extension product of the other primer (c) deoxynucleotide triphosphate (dNTP)
(D) a buffer solution containing divalent ions and monovalent ions, and (e) a MutS mutant with reduced inactivation activity against nucleoside triphosphates [10]
[8] A reagent or kit comprising the reaction solution composition according to any one of [9].

  According to the present invention, the inhibition of amplification by MutS can be avoided by preventing the inactivation of nucleoside triphosphate occurring during the preparation of the reaction solution and during the nucleic acid amplification reaction. Furthermore, the concentration range showing the nonspecific amplification reduction effect of MutS can be expanded. Therefore, it is possible to facilitate the preparation of a reagent to which MutS is added.

It is the figure which evaluated the inactivation activity with respect to the nucleoside triphosphate of Tth MutS. It is the figure which confirmed the validity of the evaluation of the inactivation activity with respect to the nucleoside triphosphate of Tth MutS. It is the figure which evaluated the inactivation activity with respect to the nucleoside triphosphate of a Tth MutS variant.

Hereinafter, the present invention will be described in detail.
One embodiment of the present invention is a method for amplifying a nucleic acid, which contains a MutS mutant with reduced inactivation activity for nucleoside triphosphates in a reaction solution.

  Examples of the amplification system used in the nucleic acid amplification method of the present invention include PCR, LAMP, TRC, NASBA and the like, but are not particularly limited. Hereinafter, the present invention will be described by taking the PCR method as an example, but application to other amplification systems is not excluded.

[1] MutS used in the nucleic acid amplification method of the present invention such as definition / evaluation method is a DNA mismatch binding protein, which recognizes and binds to a mismatch base pair.

In the present invention, the mismatch base pair means a mismatch except for the Watson-Crick base pair.

The nucleoside triphosphate in the present invention is adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), deoxyadenosine triphosphate (dATP). , Deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP), and the like. However, it is not limited to this.

The inactivating activity of MutS for nucleoside triphosphates in the present invention can be evaluated by PCR. For example, a PCR reaction solution containing template DNA, buffer material, magnesium, dNTPs, primers, DNA polymerase, and MutS (1.8 μM) to be evaluated is prepared, and this PCR reaction solution is immediately subjected to thermal cycling. This can be confirmed by comparing the amount of amplification between the sample (A) and the sample (B) in which the reaction solution is allowed to stand at 25 ° C. for 1 hour and then subjected to thermal cycling. When amplification cannot be confirmed in sample (B), or when the amount of amplification is reduced as compared with sample (A), MutS has an inactivating activity against nucleoside triphosphate, and sample If the same amount of amplification can be confirmed between (A) and sample (B), MutS does not have inactivation activity for nucleoside triphosphates.

<Evaluation method of inactivation activity of MutS for nucleoside triphosphate>
Evaluation of “inactivation activity against nucleoside triphosphates” in the present invention follows the following method.
Using Buffer, dNTPs, MgSO ₄ , enzyme solution (KOD-Plus-) attached to KOD-Plus- (Toyobo), 1 × PCR Buffer, and 1.0 mM MgSO ₄ , 15 pmol primer (SEQ ID NOs: 2 and 3) ), 20 ng of human genomic DNA (Roche), and a solution containing 20 μl containing 1 U of KOD-Plus-, and a solution containing MutS (1.8 μM) and MutS not contained in this solution Prepare a reaction solution. A sample to be subjected to PCR immediately with the reaction solution and a sample to be subjected to PCR after being allowed to stand at room temperature for 1 hour are prepared. PCR is performed in PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles at 94 ° C. for 1 minute followed by 40 cycles of 94 ° C., 20 seconds → 60 ° C., 30 seconds → 68 ° C., 4 minutes. After completion of the reaction, the amplified product is confirmed using a DNA-12000 kit of MultiNA (manufactured by Shimadzu Corporation). In the case where the MutS concentration is 1.8 μM, the inactivation activity of MutS for nucleoside triphosphates is compared by comparing the amount of amplification between the sample immediately subjected to thermal cycling and the sample subjected to thermal cycling after being left for 1 hour. Can be evaluated.
In this evaluation method, the “reaction solution not containing MutS” is used as a control for eliminating the influence on the amplification amount when left for 1 hour.

[2] Nucleic Acid Amplification Method The composition of the reaction solution composition for carrying out the nucleic acid amplification method of the present invention is particularly that the MutS mutant with reduced inactivation activity to nucleoside triphosphate is included in the reaction solution. It is not limited. For example, in the case of a PCR reaction solution, a heat-resistant DNA polymerase, a template nucleic acid, one or more oligonucleotide primers, one or more deoxynucleotide triphosphates or deoxynucleotide triphosphate derivatives, buffers, and salts What is necessary is just to contain at least 1 among the groups which consist.

In the case of performing PCR as a nucleic acid amplification method, examples of the reaction solution composition include the following (a) to (e), but are not limited thereto.
(A) DNA polymerase (b) a pair of primers in which one primer is complementary to the DNA extension product of the other primer (c) deoxynucleotide triphosphate (dNTP)
(D) a buffer solution containing a divalent ion and a monovalent ion, and (e) a MutS mutant with reduced inactivation activity against nucleoside triphosphates. These reagents may include, for example, PCR enhancing factors, BSA, nonionic surfactants, and the like.

The reaction solution having the above configuration can be in the form of a reagent or a kit. The reagent or kit for carrying out the nucleic acid amplification method of the present invention is characterized in that it contains a MutS mutant with reduced inactivation activity against nucleoside triphosphates, and other configurations are not particularly limited. For example, all the reaction liquid compositions may be combined into one liquid, or may be divided into two or more liquids.

  The thermostable DNA polymerase used in the method of the present invention is not particularly limited. Conventionally known polymerases derived from thermostable bacteria can be used. Specifically, Taq DNA polymerase and Tth DNA polymerase belonging to family A (PolI type), KOD DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, Ultimate DNA polymerase, PrimeSTAR (registered trademark) belonging to family B (α type) Examples include DNA polymerase series (HS, GXL, Max). Two or more of these may be used in combination.

  In the method of the present invention, the nucleic acid used as a template is not particularly limited. Examples include synthetic DNA, genomic DNA purified from biological samples, and cDNA obtained from RNA by reverse transcription. Further, there is no need for purification, and a crude sample of a biological sample containing a nucleic acid as a template or the biological sample itself can be used.

The one or more oligonucleotide primers used in the present invention are oligonucleotides complementary to each nucleic acid strand of the nucleic acid to be amplified, and it is preferable to use two or more primers. The primer is substantially complementary to each strand of a double-stranded nucleic acid, and when the extension product synthesized from one primer is separated from its complement, the extension product is It is chosen so that it can serve as a template for the synthesis of the extension product of the other primer.

  One or more deoxynucleotide triphosphates or derivatives of deoxynucleotide triphosphates are used as substrates in PCR reactions. As this substrate, four kinds of deoxyribonucleoside triphosphate (dNTPs; a mixture of dATP, dCTP, dTTP and dGTP) solution and the like may be contained.

The PCR reaction solution preferably further contains a buffer. Examples of the buffer include Tris (TRIS), Tricine (TRICINE), Bis-Tricine (BIS-TRICINE), Hepes (HEPES), Mops (MOPS), Tes (TES), Tapes (TAPS), Pipes (PIPES). ), And caps (CAPS), but are not particularly limited. As a density | concentration, about 10-200 mM is preferable and about 20-100 mM is more preferable. As pH, the range of about 7.0-9.5 is preferable, and the range of about 7.5-9.0 is more preferable.
The buffer solution preferably contains Mg ²⁺ at a concentration of about 1 to 5 mM, preferably about 1.5 to 2.5 mM. Furthermore, KCl may be included.

  Furthermore, the PCR reaction solution may contain albumin, glycerol, heparin, trehalose, betaine and the like. What is necessary is just to add these addition ratios in the range which does not inhibit PCR reaction.

[3] MutS mutant The origin of MutS before mutation of the MutS mutant used in the present invention is not particularly limited.
MutS is derived from MutS homologs 2-6 (SEQ ID NOs: 4-8), Escherichia coli (SEQ ID NO: 9), Salmonella typhimurium (SEQ ID NO: 10), Streptococcus hexA (SEQ ID NO: 11), and Thermos aquaticus (Thermus aquaticus). (SEQ ID NO: 12), Aquifex aeolicus (SEQ ID NO: 13), Thermus thermophilus (Tth) (SEQ ID NO: 1), and the like, but are not limited thereto. In addition, Thermococcus kodakaraensis (SEQ ID NO: 14) and Pyrococcus furiosus (SEQ ID NO: 15), which are hyperthermophilic bacteria, can be used, but other Pyrococcus and Thermococcus can also be used. Taq, Aquifex aeolicus and Tth are preferred.

  The MutS mutant used in the present invention is not particularly limited as long as it is modified so as to reduce the inactivation activity against nucleoside triphosphates.

There are no particular limitations on the mutation site where such a change in properties can be obtained, but a mutation in the ATPase active site is preferred. For example, Taq MutS described in Non-Patent Document 5 corresponds to amino acids 543 to 731 in the amino acid sequence of SEQ ID NO: 12.
The ATPase active site is not specified except for MutS derived from Taq, but the amino acids related to the ATPase active site are highly conserved in MutS of different origin (Non-Patent Documents 2 to 4), and the primary structure of the amino acid sequence By comparing (aligning), the position can be predicted and confirmed with high probability.

In the present specification, in a amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1, a position (order) on SEQ ID NO: 1 and a corresponding position are when the primary structure of the sequence is compared (alignment) , A position corresponding to the position of SEQ ID NO: 1.
In this specification, by using default (initial setting) parameters in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php/lang=ja) of DNA Database of Japan (DDBJ), Compare the primary structure of the sequences.

The present inventors have confirmed by the above method that the ATPase active site of Tth-derived MutS means a portion corresponding to amino acids 551 to 739 in the amino acid sequence set forth in SEQ ID NO: 1. The same confirmation can be made with a MutS other than the MutS whose sequence is specifically presented in this specification.

In the nucleic acid amplification method of the present invention, the MutS mutant with reduced inactivation activity for nucleoside triphosphates is modified from a polar amino acid residue in the ATPase active site to a nonpolar amino acid residue or an amino acid residue having the opposite polarity Preferably, the MutS mutant.
Polar amino acids in the present invention are aspartic acid, asparagine, glutamic acid, glutamine, cysteine, tyrosine, arginine, histidine, lysine, threonine, and serine, and nonpolar amino acids are glycine, alanine, valine, proline, phenylalanine, leucine, Isoleucine, tryptophan, and methionine.

In the nucleic acid amplification method of the present invention, the MutS mutant with reduced inactivation activity against nucleoside triphosphate is more preferably at least one amino acid selected from amino acids corresponding to amino acids K597, D670, and E671 in SEQ ID NO: 1. With modifications.
In a preferred example, the K597 amino acid has lysine (K) replaced with a nonpolar amino acid, specifically a K597M or K597A amino acid substitution. In another preferred example, the D670 amino acid has aspartic acid (D) replaced with a nonpolar amino acid, specifically an amino acid substitution of D670A. In another preferred example, the E671 amino acid is substituted with an amino acid having an opposite polarity or a nonpolar amino acid in glutamic acid (E), specifically an amino acid substitution of E671K, E671A, E671M, or E671N.

Here, for example, K597 means a lysine (K) residue which is the 597th amino acid, and one letter of the alphabet represents an abbreviation of a commonly used amino acid. In the present specification, for example, K597M means that lysine (K) of the 597th amino acid is replaced with methionine (M), and so on.

[4] Method for producing MutS mutant A method for modifying MutS used in the nucleic acid amplification method of the present invention has already been established in the art. Therefore, it can modify | change according to a well-known method, The aspect in particular is not restrict | limited.

As one embodiment of a method for introducing an amino acid modification, a site-directed mutagenesis method based on the inverse PCR method can be used. For example, KOD-Plus-Mutageness Kit (manufactured by Toyobo) (1) denatures a plasmid into which a target gene has been inserted, anneals the mutation primer to the plasmid, and then performs an extension reaction using KOD DNA polymerase. (2) Repeat the cycle of (1) 15 times, (3) Selectively cleave only the plasmid using the restriction enzyme DpnI, (4) Phosphorylation of newly synthesized gene, Ligation (5) A kit that can transform a cyclized gene into Escherichia coli and obtain a transformant having a plasmid introduced with the target mutation.

  The above-mentioned modified MutS gene is transferred to an expression vector as necessary. For example, Escherichia coli as a host is transformed with the expression vector, and then applied to an agar medium containing a drug such as ampicillin to form a colony. The colony is inoculated in a nutrient medium such as LB medium or 2 × YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted. A vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. This crude enzyme solution is heat-treated at 70 ° C. for 30 minutes, centrifuged to remove host-derived protein, and subjected to SDS-PAGE, thereby confirming the expression of the target protein.

  Any method may be used as a method for obtaining purified MutS from the strain selected by the above method, for example, the following method. After collecting the cells obtained by culturing in a nutrient medium, the crude enzyme solution is obtained by crushing and extraction by enzymatic or physical crushing methods. The obtained crude enzyme extract can be heat-treated, for example, at 70 ° C. for 30 minutes, and purified by a metal affinity method using HisTrap HP (manufactured by GE Healthcare). After this operation, MutS can be purified by subjecting it to phenyl sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to such an extent that it shows an almost single band.

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1: Preparation of Tth MutS A plasmid containing the MutS (Tth MutS) gene derived from Thermus thermophilus was prepared.
The Thermus thermophilus gene expression plasmid was provided by RIKEN BioResource Center through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology (Non-patent Document 6). Using this, the sequence of TthMutS was cloned into pBluescript. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.

The cells obtained in Example 1 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / ml ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated at 37 ° C. for 16 hours in 3 ml of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / ml ampicillin in advance. Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution by centrifugation, suspended in 15 ml of disruption buffer (50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 5 mM imidazole), and then disrupted by sonication. A cell lysate was obtained. Next, the cell lysate was treated at 70 ° C. for 30 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, Ni affinity chromatography and phenyl sepharose chromatography were performed, and finally, the solution was replaced with a storage buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA) to obtain Tth MutS.

Example 2: Evaluation of inactivation activity of Tth MutS for nucleoside triphosphate As a method of evaluating the inactivation activity of Tth MutS for nucleoside triphosphate, after preparing a reaction solution containing MutS, it was allowed to stand at room temperature for 1 hour. The difference in the amount of amplification by PCR was compared by amplifying 3.6 kb of Human β-globin.

PCR was performed using Buffer, dNTPs, MgSO ₄ , and enzyme solution (KOD-Plus-) attached to KOD-Plus- (Toyobo), 1 × PCR Buffer, and 1.0 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 2 and 3), 20 ng of human genomic DNA (Roche), and 20 μl containing 1 U of KOD-Plus- were prepared, and then MutS (0.5, 0.8, 1.0 , 1.3, 1.5 and 1.8 μM) and a reaction solution not containing MutS were prepared. A sample to be subjected to PCR immediately with the reaction solution and a sample to be subjected to PCR after being allowed to stand at room temperature for 1 hour were prepared. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles of 94 ° C, 20 seconds → 60 ° C, 30 seconds → 68 ° C, 4 minutes after a previous reaction at 94 ° C for 1 minute. After completion of the reaction, the amplified product was confirmed using a DNA-12000 kit of MultiNA (manufactured by Shimadzu Corporation).

The results are shown in FIG. At a Tth MutS concentration of 1.8 μM, amplification was confirmed in the immediately following sample, but amplification was not confirmed in the sample left at room temperature for 1 hour. From this, it was confirmed that Tth MutS has an inactivating activity for nucleoside triphosphates.

Example 3: Confirmation of evaluation method of inactivation activity for nucleoside triphosphates It was confirmed whether the cause of the inhibition of amplification was due to the inactivation activity for nucleoside triphosphates. As an evaluation method, a reaction solution excluding dNTPs was prepared and allowed to stand at room temperature for 1 hour, and then PCR was performed by adding dNTPs immediately before PCR. The difference in the amount of amplification between immediately after and 1 hour after PCR was compared by amplifying 3.6 kb of Human β-globin.

PCR was performed using Buffer, MgSO ₄ , enzyme solution (KOD-Plus-) attached to KOD-Plus- (Toyobo), 1 × PCR Buffer, and 1.0 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 2 and 3), a solution containing 20 ng of human genomic DNA (Roche) and 1 U of KOD-Plus- containing 20 μl excluding dNTPs, and further adding MutS (0.5, 0.8, 1.0, 1.3, 1.5 and 1.8 μM) and a reaction solution not containing MutS were prepared. A sample in which PCR was performed immediately after adding 0.2 mM dNTP in the reaction solution, and a sample in which 0.2 mM dNTPs was added immediately before performing PCR after standing at room temperature for 1 hour were prepared. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles of 94 ° C, 20 seconds → 60 ° C, 30 seconds → 68 ° C, 4 minutes after a previous reaction at 94 ° C for 1 minute. After completion of the reaction, the amplified product was confirmed using a DNA-12000 kit of MultiNA (manufactured by Shimadzu Corporation).

The results are shown in FIG. At a Tth MutS concentration of 1.8 μM, amplification could be confirmed in both the immediately following sample and the sample left at room temperature for 1 hour, and amplification inhibition was not confirmed. From this, it was confirmed that the cause of the inhibition of Tth MutS amplification was the inactivation activity on nucleoside triphosphate, and the validity of the evaluation method was shown.

Example 4: Preparation of TthMutS mutant A plasmid containing a modified MutS (Tth MutS) gene derived from Thermus thermophilus was prepared.
The DNA template used for mutagenesis was MutS (Tth MutS) derived from Thermus thermophilus cloned in pBluescript. The mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.
The plasmid prepared in Example 4 is shown in Table 1.

  Culture of the bacterial cells obtained in Example 4 was performed as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / ml ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated at 37 ° C. for 16 hours in 3 ml of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / ml ampicillin in advance. Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution by centrifugation, suspended in 15 ml of disruption buffer (50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 5 mM imidazole), and then disrupted by sonication. A cell lysate was obtained. Next, the cell lysate was treated at 70 ° C. for 30 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, Ni affinity chromatography was performed, and finally, a storage buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA) was substituted to obtain a MutS mutant.

Example 5: Evaluation of inactivation activity of Tth MutS mutant for nucleoside triphosphates Evaluation method for inactivation activity of Tth MutS mutants (K597M, K697A, D670A, E671A, E671M, E671K, E671N) against nucleoside triphosphates After preparing the reaction solution, the reaction solution was allowed to stand at room temperature for 1 hour, and the difference in amplification amount by PCR was compared by amplifying 3.6 kb of human β-globin.

PCR was performed using Buffer, dNTPs, MgSO ₄ , and enzyme solution (KOD-Plus-) attached to KOD-Plus- (Toyobo), 1 × PCR Buffer, and 1.0 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 2 and 3), a solution containing 20 μl containing 20 ng human genomic DNA (Roche) and 1 U KOD-Plus-, and a reaction containing MutS (1.0 and 1.8 μM) in this solution A solution and a reaction solution not containing MutS were prepared. A sample to be subjected to PCR immediately with the reaction solution and a sample to be subjected to PCR after being allowed to stand at room temperature for 1 hour were prepared. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles of 94 ° C, 20 seconds → 60 ° C, 30 seconds → 68 ° C, 4 minutes after a previous reaction at 94 ° C for 1 minute. After completion of the reaction, the amplification product was confirmed using a DNA-12000 kit of MultiNA (manufactured by Shimadzu Corporation).

The results are shown in FIG. At a TthMutS concentration of 1.8 μM, amplification could be confirmed in both the immediately following sample and the sample left at room temperature for 1 hour, and no amplification inhibition was confirmed. From this, since the reaction liquid was stable at room temperature, preparation became easy.

  The present invention eliminates the inhibition of amplification due to the addition of MutS in the nucleic acid amplification reaction, and improves the effect of suppressing the nonspecific amplification reaction of MutS. As a result, not only in the research field but also in the forensic field, foods, environmental microbiological tests, diagnostic applications, etc., it becomes possible to easily confirm amplification of only the target product.

Claims (9)

A nucleic acid amplification method comprising, in a reaction solution, a MutS mutant that has a reduced inactivation activity against nucleoside triphosphates and has a modified ATPase active site. The nucleic acid amplification method according to claim 1, wherein the MutS mutant is a MutS mutant in which a polar amino acid residue of an ATPase active site is modified to a nonpolar amino acid residue or an amino acid residue having an opposite polarity. The MutS variant is a MutS variant having a modification of at least one amino acid among amino acids related to the ATPase active site consisting of amino acids corresponding to the 597th, 670th and 671th amino acids in the amino acid shown in SEQ ID NO: 1. The method according to 1 or 2. The nucleic acid amplification method according to claim 3, wherein MutS, which is an amino acid substitution of K597M or K597A, is used as the modification corresponding to K597. The nucleic acid amplification method according to claim 3, wherein MutS in which the modification corresponding to D670 is an amino acid substitution of D670A is used. The nucleic acid amplification method according to claim 3 , wherein MutS, which is an amino acid substitution of E671A, E671M, E671K or E671N, is used as the modification corresponding to E671. A reaction solution composition for use in the nucleic acid amplification method according to claim 1. The reaction liquid composition of Claim 7 containing the following (a)-(e).
(A) DNA polymerase (b) a pair of primers in which one primer is complementary to the DNA extension product of the other primer (c) deoxynucleotide triphosphate (dNTP)
(D) a buffer solution containing divalent ions and monovalent ions, and (e) a MutS mutant with reduced inactivation activity against nucleoside triphosphates A reagent or kit comprising the reaction solution composition according to claim 7 or 8.