Heat resistant mismatched endonuclease variants

Abstract

The present invention provides heat resistant mismatched endonuclease variants. In particular, the present invention provides GG-specific mismatched endonuclease variants. The invention also provides a mismatch-specific cleavage reaction using the variant, a method of removing errors in a nucleic acid amplification reaction using a mismatch nuclease, a method of inhibiting amplification of a nucleic acid having a specific base sequence during a nucleic acid amplification reaction, and a method of inhibiting amplification of a nucleic acid having a specific base sequence during a nucleic acid amplification reaction. And a method for detecting a nucleic acid having a single base polymorphism mutation using the inhibition method.

Description

This application is a divisional application of a patent application with the original application date of March 18, 2021, application number 202180021831.5 (international application number PCT / JP2021 / 011033), and invention name “Thermoresistant mismatch endonuclease variant”. Technical Field

[0001] The present invention relates to a thermostable mismatch endonuclease mutant that recognizes and cleaves mismatch base pairs (hereinafter also referred to as mismatches) in double-stranded nucleic acids, a composition comprising the mismatch endonuclease mutant, and a method of using the mismatch endonuclease mutant. Background Art

[0002] In recent years, biotechnology has achieved remarkable development. In particular, due to large-scale genome analysis, with the progress of genome analysis technology, a large amount of genome sequence information has been accumulated. In addition, based on the combination of the above information and various physiological function analyses, many functional gene mutations have been found. The analysis of these mutations has been used for human gene diagnosis, as well as the improvement of crops and the separation or manufacture of useful microorganisms, and has therefore made a great contribution to general life.

[0003] Mutation analysis is performed by direct analysis of the genomic sequence or by using an enzyme that recognizes mismatched base pairs. Mutation analysis methods include the use of detection of factors that can specifically bind to mismatched base pairs formed by the pairing of mutant DNA with wild-type DNA. Representative examples of mutation analysis methods include detection of mutation sites using MutS, MutT and MutL complexes from Escherichia coli (Patent Document 1).

[0004] The mutation analysis method including the mismatch endonuclease using specific cutting mismatch site is also known. In this method, the mismatch endonuclease is used to cut DNA near the mismatch base pair, and the DNA fragment obtained thereby is analyzed to detect whether there is a mutation and the position of the mutation. As a representative example, it is known to use the method (patent documentation 2) of the cell gene product from celery, and the method is actually used for the analysis of base mutation. However, the enzyme is not heat-resistant, and therefore can not be used for the technology related to the high temperature reaction process, such as PCR. Therefore, in order to detect base mutation, the method needs four steps of amplification, mismatch formation, mismatch cutting and detection.

[0005] Other examples of mismatch endonucleases include mismatch endonucleases from Pyrococcus furiosus, Methanocaldococcus jannaschii, Thermococcus barophylus, and Thermococcuskodakarensis (Patent Documents 3 and 4). However, these mismatch endonucleases recognize and cut multiple mismatches, and therefore have a problem of mismatch recognition (substrate specificity).

[0006] In some cases, the substrate specificity of the mismatched endonuclease is not suitable for the gene mutation to be detected, and therefore the gene mutation cannot be analyzed. In this case, except that the appropriately designed nucleic acid (so-called inhibitory oligonucleotide) is added to the reaction system to specifically detect the gene mutation, there is no other choice (patent documentation 5).

[0007] In addition to mutation analysis, examples of biotechnology techniques with great influence include nucleic acid amplification techniques. A representative example of nucleic acid amplification techniques is polymerase chain reaction (PCR). PCR is a technique for easily amplifying desired nucleic acid fragments in vitro. PCR is an experimental technique that is indispensable in the fields including biology, medicine and agriculture and in a wide range of fields for research on genes. PCR is also applied to the detection of mutant genes and the analysis of DNA methylation. Isothermal nucleic acid amplification methods, such as LAMP method and ICAN method, do not require special equipment and are therefore used as cheaper nucleic acid detection methods. For the whole genome structure analysis carried out in recent years, the whole genome amplification method is an important technology, particularly for the analysis of rare samples.

[0008] In these nucleic acid amplification methods, the incorporation of an erroneous base occurs with a constant probability. The probability has been reduced by improvements in polymerases, etc. However, the incorporation of an erroneous base still interferes with accurate analysis.

[0009] Nucleic acid amplification technology is not only used to amplify DNA with a specific nucleotide sequence, but also to amplify a mixture of DNA with a common nucleotide sequence region at both ends. Specific examples of this nucleic acid amplification technology include the construction of a genomic library or a cDNA library. However, in the construction of such libraries, DNA molecules with higher content are preferentially amplified, which may interfere with the analysis or screening of various DNAs.

[0010] In order to solve the above problem, the ratio of DNA with higher content is reduced by utilizing the normalization of self-hybridization (non-patent literature 1). SSH-PCR (non-patent literature 2) combining PCR and self-hybridization is also used. However, using these methods, DNA homologous to the DNA with higher content is also removed.

[0011] In DNA detection by nucleic acid amplification methods, target DNA and non-target DNA may compete for amplification. In other words, when amplifying non-target DNA while amplifying target DNA, it is difficult to detect target DNA. The above problem can be solved by using real-time PCR, in which probes such as cycling probes or TaqMan probes are used to detect only target DNA. However, in the case where non-target DNA is excessively present in large quantities relative to target DNA, it is difficult to detect target DNA due to false positive reactions with non-target DNA.

[0012] Such problems may arise, for example, in detecting small numbers of mutant alleles in the presence of normal alleles (e.g., detecting circulating tumor genes), detecting small numbers of methylated or unmethylated alleles by epigenetic analysis, detecting small numbers of fetal DNA sequences circulating in the mother's blood, etc.

[0013] In order to solve the above problems, a method called enrichment PCR or restriction endonuclease-mediated selective polymerase chain reaction (REMS PCR) has been developed (non-patent document 3). This method involves the use of a thermostable restriction enzyme. In this method, a primer is used to selectively detect a DNA having a mutant nucleotide sequence, and the primer is, for example, designed so that the cutting of the restriction enzyme occurs only when the template has a normal nucleotide sequence. However, depending on the target DNA to be detected, there may not be a thermostable restriction enzyme with a recognition sequence suitable for detection by REMS PCR.

[0014] Reference List Patent Literature Patent document 1: US 5922539 B Patent document 2: WO 01 / 062974 Patent Document 3: WO 2014 / 142261 Patent Document 4: WO 2016 / 039377 Patent Document 5: WO 2016 / 152812 Non-patent literature Non-patent document 1: "Nucleic Acids Research", February 2004, Vol. 32, No. 3, e37 Non-patent document 2: "Methods in Molecular Biology", 2009, Vol. 496, No. 2, pp. 223-243 Non-patent document 3: "American Journal of Pathology", August 1998, Vol. 153, No. 2, pp. 373-379. Summary of the invention

[0015] Technical issues Objects of the present invention include providing a thermostable mismatch endonuclease mutant having improved mismatch specificity compared to conventional mismatch endonucleases, a composition comprising the mismatch endonuclease mutant, and a method of using the mismatch endonuclease mutant.

[0016] Solutions to the problem Under the above circumstances, through in-depth efforts, the inventors successfully produced a mismatch endonuclease mutant capable of specifically cutting a GG mismatch and a mismatch endonuclease mutant capable of specifically cutting a TT mismatch. In addition, although the mismatch endonuclease was originally a homodimer, the inventors successfully connected two monomers from the above two mutants with a linker, thereby producing a mismatch endonuclease mutant capable of specifically cutting a GT / TG mismatch. Thus the present invention was completed.

[0017] Specifically, the first aspect of the present invention provides: [1] A polypeptide represented by the following (A) or (B): (A) A polypeptide selected from the group consisting of the following (1) to (4) and having an activity of cleaving a nucleic acid chain having guanine (G) that forms a mismatched base pair in a double-stranded nucleic acid: (1) A polypeptide comprising an amino acid sequence comprising, in the amino acid sequence shown in SEQ ID NO: 1, substitution of serine at position 47 with a basic amino acid residue and substitution of asparagine at position 76 with an acidic amino acid residue; (2) A polypeptide comprising an amino acid sequence comprising a mutation of an additional 1 to 10 amino acid residues in the substituted amino acid sequence described in (1), wherein the mutation is a substitution, deletion, insertion and / or addition, and the amino acid substitution in the substituted amino acid sequence described in (1) is retained; (3) a polypeptide comprising an amino acid sequence having 50% or more homology with the substituted or mutated amino acid sequence described in (1) or (2), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (1) or (2) is retained; and (4) a polypeptide comprising an amino acid sequence having 50% or greater identity to the substituted or mutated amino acid sequence described in (1) or (2), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (1) or (2) is retained; or (B) A polypeptide selected from the group consisting of the following (5) to (8) and having an activity of cleaving a nucleic acid chain having thymine (T) that forms a mismatched base pair in a double-stranded nucleic acid: (5) A polypeptide comprising an amino acid sequence comprising, in the amino acid sequence shown in SEQ ID NO: 1, substitution of glutamine at position 78 by a basic amino acid residue and substitution of leucine at position 123 by a neutral (polar, uncharged) amino acid residue or a sulfur-containing amino acid residue, or substitution of leucine at position 125 by a hydrophobic amino acid residue or a sulfur-containing amino acid residue; (6) A polypeptide comprising an amino acid sequence comprising a mutation of an additional 1 to 10 amino acid residues in the substituted amino acid sequence described in (5), wherein the mutation is a substitution, deletion, insertion and / or addition, and the amino acid substitution in the substituted amino acid sequence described in (5) is retained; (7) A polypeptide comprising an amino acid sequence having 50% or more homology with the substituted or mutated amino acid sequence described in (5) or (6), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (5) or (6) is retained; and (8) A polypeptide comprising an amino acid sequence having 50% or higher identity with the substituted or mutated amino acid sequence described in (5) or (6), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (5) or (6) is retained; [2] The polypeptide according to [1], wherein: The basic amino acid residue is lysine, arginine or histidine, The acidic amino acid residue is aspartic acid or glutamic acid, The neutral (polar, uncharged) amino acid is threonine, serine, asparagine, glutamine, tyrosine or cysteine, The sulfur-containing amino acid is cysteine ​​or methionine, and The hydrophobic (non-polar) amino acid is alanine, glycine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine or methionine; [3] The polypeptide according to [1] or [2], comprising an amino acid sequence selected from SEQ ID NOs: 6 to 15; [4] A nucleic acid encoding the polypeptide according to any one of [1] to [3]; [5] The nucleic acid according to [4], comprising a nucleotide sequence selected from SEQ ID NOs: 21 to 30; [6] An expression vector comprising the nucleic acid according to [4] or [5] and an expression control sequence; [7] A cell transformed with the expression vector according to [6], which expresses a polypeptide having mismatch endonuclease activity; [8] A method for cleaving a double-stranded nucleic acid having a mismatch, the method comprising the step of treating the double-stranded nucleic acid with the polypeptide according to any one of [1] to [3]; [9] A composition comprising the following (a) to (d): (a) the polypeptide according to any one of [1] to [3]; (b) an oligonucleotide which, when hybridized with a non-target nucleic acid, forms a double-stranded nucleic acid comprising at least one mismatch, wherein the double-stranded nucleic acid is cleaved by the polypeptide of (a), and which, when hybridized with a target nucleic acid, forms a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having DNA polymerase activity;

[10] A method for amplifying a nucleic acid, the method comprising the following steps (1) and (2): (1) A step of preparing a composition comprising a nucleic acid molecule as a template and the following (a) to (d): (a) the polypeptide according to any one of [1] to [3]; (b) an oligonucleotide which, when hybridized with a non-target nucleic acid, forms a double-stranded nucleic acid comprising at least one mismatch, wherein the double-stranded nucleic acid is cleaved by the polypeptide of (a), and which, when hybridized with a target nucleic acid, forms a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having DNA polymerase activity; and (2) a step of reacting the composition obtained in step (1) under appropriate conditions to perform nucleic acid amplification;

[11] The method according to

[10] , wherein the nucleic acid amplification is performed by a polymerase chain reaction (PCR) method, an isothermal nucleic acid amplification method or a multiple displacement amplification (MDA) method;

[12] A method for inhibiting amplification of a nucleic acid containing a specific nucleotide sequence, the method comprising the step of performing a nucleic acid amplification reaction in the presence of the following (a) to (d): (a) an oligodeoxynucleotide designed to produce one or several mismatches when hybridizing with a nucleic acid comprising a specific nucleotide sequence or its complementary strand; (b) DNA polymerase; (c) at least one pair of oligonucleotide primers; and (d) the polypeptide according to any one of [1] to [3];

[13] The method according to

[12] , wherein the nucleic acid amplification reaction is polymerase chain reaction (PCR) or isothermal nucleic acid amplification;

[14] A method for preferentially amplifying a target DNA, the method comprising inhibiting amplification of a DNA having a nucleotide sequence that differs from the target DNA by one or several nucleotides by the method according to

[12] or

[13] ;

[15] The method according to

[14] , which is used to amplify one of a DNA having a wild-type nucleotide sequence and a DNA that differs from the wild-type DNA by a single nucleotide polymorphism mutation that is different from each other;

[16] The method according to

[15] , wherein the single nucleotide polymorphism mutation is a single nucleotide polymorphism mutation associated with carcinogenesis or a therapeutic effect of a cancer therapeutic agent; and

[17] A polypeptide in dimer form selected from the group consisting of: (1) a homodimer comprising the polypeptide according to claim 1(A) as a subunit, (2) a homodimer having the polypeptide according to claim 1(B) as a subunit, and (3) A heterodimer comprising the polypeptide according to [1] (A) and the polypeptide according to [1] (B) as subunits.

[0018] Effects of the Invention According to the present invention, there are provided a thermostable mismatch endonuclease mutant having great utility in biotechnology, a composition comprising the mismatch endonuclease mutant, and a method of using the mismatch endonuclease mutant.

[0019] Description of the implementation plan As used herein, the term "mismatch" refers to base pairing other than Watson-Crick base pairs present in double-stranded nucleic acids, in other words, base pairing other than a combination of base pairings of G (guanine base)-C (cytosine base), and A (adenine base)-T (thymine base) or U (uracil base).

[0020] As used herein, the term "GG-specific" refers to specificity for GG mismatches, the term "TT-specific" refers to specificity for TT mismatches, and the term "GT / TG-specific" refers to specificity for GT or TG mismatches. In other words, these terms mean that substantially only the mismatches indicated are recognized.

[0021] As used herein, the term "polypeptide with mismatch endonuclease activity (sometimes referred to as mismatch endonuclease)" refers to a nuclease having the activity of cutting mismatch sites present in double-stranded nucleic acids. The mismatch endonuclease activity includes the activity of cutting phosphodiester bonds near the nucleotides forming mismatch base pairs, and the activity of cutting phosphodiester bonds near the nucleotides located 1-5 base pairs, preferably 1-3 base pairs away from the mismatch base pairs. In the present invention, the mismatch endonuclease is preferably a nuclease having the activity of specifically recognizing a specific mismatch base pair in a double-stranded nucleic acid to cut the double-stranded nucleic acid. As used herein, a thermostable mismatch endonuclease refers to a nuclease having the activity of cutting mismatch sites present in a double-stranded nucleic acid at a temperature of 50°C or higher. In the present invention, a thermostable mismatch endonuclease is preferably used.

[0022] The mismatch endonuclease in the present invention is an enzyme that forms a dimer and exerts its activity. It is known that when the monomers of the protein (also referred to as "subunits", meaning components of dimers) are expressed in hosts such as Escherichia coli, the monomers naturally combine to form dimers (homodimers). As used herein, "a polypeptide having the activity of cutting a nucleic acid chain, wherein the nucleic acid chain has guanine (G) that forms a mismatch base pair in a double-stranded nucleic acid" and "a polypeptide having the activity of cutting a nucleic acid chain, wherein the nucleic acid chain has thymine (T) that forms a mismatch base pair in a double-stranded nucleic acid" refer to monomers.

[0023] Amino acids are divided into hydrophilic (polar) amino acids and hydrophobic (non-polar) amino acids according to their chemical properties.

[0024] Hydrophilic (polar) amino acids include acidic amino acids, basic amino acids, neutral (polar, uncharged) amino acids, tyrosine among aromatic ring-containing amino acids, and cysteine ​​among sulfur-containing amino acids.

[0025] The hydrophobic (non-polar) amino acids include amino acids with aliphatic side chains, tryptophan and phenylalanine among the aromatic ring-containing amino acids, and methionine among the sulfur-containing amino acids.

[0026] Acidic amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E).

[0027] Basic amino acids include lysine (Lys, K), arginine (Arg, R), and histidine (His, H).

[0028] Neutral (polar, uncharged) amino acids include threonine (Thr, T), serine (Ser, S), asparagine (Asn, N), glutamine (Gln, Q), tyrosine (Tyr, Y), and cysteine ​​(Cys).

[0029] Amino acids containing aromatic rings include tyrosine (Tyr, Y), tryptophan (Trp, W), and phenylalanine (Phe, F).

[0030] Sulfur-containing amino acids include cysteine ​​(Cys, C) and methionine (Met, M).

[0031] Amino acids with aliphatic side chains include alanine (Ala, A), glycine (Gly, G), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I) and proline (Pro, P). Proline is an imino acid with a cyclized side chain.

[0032] As used herein, "similar amino acids" refer to amino acids that are classified into the same group based on the above-mentioned chemical properties. For example, lysine, arginine, and histidine are classified as basic amino acids and are therefore amino acids that are similar to each other.

[0033] As used herein, "amino acid sequence homology" refers to the homology or similarity between polypeptide sequences taking into account the above-mentioned similar amino acids. As described above, amino acids classified into the same group based on the chemical properties of amino acids are considered to be "homologous".

[0034] As used herein, "amino acid sequence identity" refers to the identity between polypeptide sequences without considering the above-mentioned similar amino acids. As used herein, "nucleotide sequence identity" refers to the identity between polynucleotide sequences.

[0035] Known programs can be used to calculate scores for amino acid sequence homology, amino acid sequence identity, and nucleotide sequence identity. Examples of such programs include, but are not limited to, BLAST, BLAT, FASTA, SSEARCH, and MPsrch.

[0036] As used herein, amino acid numbers (also referred to as amino acid positions) are based on the amino acid sequence of SEQ ID NO: 1. Therefore, when counting from the N-terminus in a variant protein of a homologous protein derived from other organisms or a polypeptide of SEQ ID NO: 1, the amino acid position indicated by the amino acid number as used herein may be different from the amino acid position indicated by the same amino acid number. In other words, as used herein, "the position corresponding to position 47 in the amino acid sequence of the wild-type mismatch endonuclease" refers to an amino acid residue that is considered to be in the same position as position 47 in the wild-type amino acid sequence (SEQ ID NO: 1) when the wild-type amino acid sequence is compared and aligned with a mutant amino acid sequence or an amino acid sequence of a mismatch endonuclease derived from other organisms.

[0037] As used herein, "target nucleic acid" or "target DNA" is a nucleic acid that is not cut by the mismatch endonuclease mutant of the present invention. In other words, the target nucleic acid or target DNA is a nucleic acid (as a template) to be amplified by the amplification method of the present invention. On the other hand, "non-target nucleic acid" or "non-target DNA" is a nucleic acid cut by the mismatch endonuclease mutant of the present invention. In other words, non-target nucleic acid or non-target DNA is a nucleic acid that is cut by the cutting method of the present invention and is therefore not amplified by the amplification method of the present invention. "Target nucleic acid" or "target DNA" and "non-target nucleic acid" or "non-target DNA" are not particularly limited and can be any nucleic acid that is desired to be distinguished from each other.

[0038] Hereinafter, the present invention will be described in detail.

[0039] 1. Mismatched endonuclease mutants of the present invention A first aspect of the present invention relates to mismatch endonuclease mutants, i.e., mismatch endonuclease mutants with altered substrate specificity. The mismatch endonuclease mutants are characterized by changes in the amino acid sequence of the site contributing to substrate recognition and / or cleavage in the wild-type mismatch endonuclease from Pyrococcus furiosus (also known as PfuNucS) or its homologue or a mutant having the mismatch endonuclease activity of PfuNucS.

[0040] The mismatch nuclease mutants of the present invention can be prepared, for example, by introducing amino acid substitutions into the polypeptide PF_RS00065 from Pyrococcus furiosus (RefSeq ID: WP_11011124.1, SEQ ID NO: 1, formerly known as PF0012, formerly RefSeq ID: NP_577741), wherein the amino acid substitutions are (i) an amino acid substitution of serine at position 47 by a basic amino acid, and an amino acid substitution of asparagine at position 76 by an acidic amino acid, or (ii) an amino acid substitution of glutamine at position 78 by a basic amino acid, and an amino acid substitution of leucine at position 123 by a neutral (polar, uncharged) amino acid or a sulfur-containing amino acid, or an amino acid substitution of leucine at position 125 by a hydrophobic amino acid or a sulfur-containing amino acid.

[0041] In addition, the mismatch nuclease mutant of the present invention can also be prepared by introducing a mutation corresponding to the above-mentioned amino acid substitution (i) or (ii) as follows: polypeptide W77F (SEQ ID NO: 2), wherein the tryptophan at position 77 in PF_RS00065 is replaced with phenylalanine; polypeptide MJ RS01180 from Methanococcus jannaschii (Ref Seq ID: NP_247194, SEQID NO: 3, formerly known as MJ_0225), which is a homologue of PF_RS00065; polypeptide TERMP_01877 from Thermococcus barophilus (Ref Seq ID: YP_004072075, SEQ ID NO: 4), which is a homologue of PF_RS00065; or a polypeptide from Thermococcus kodakarensis strain KOD1 (JCM 12380T) (SEQ ID NO: 5), which is a homologue of PF_RS00065.

[0042] The mismatched endonuclease mutant of the present invention is preferably a thermostable enzyme. For example, a polypeptide that stably maintains activity even in a thermal cycle (such as PCR) is preferably used in the method of the present invention. In the method of the present invention, a polypeptide that is not inactivated even at 50°C or higher, preferably 60°C or higher, further preferably 70°C or higher, and more preferably 70°C or higher can be used.

[0043] For example, the mismatch endonuclease mutant of the present invention comprises the polypeptide shown in (A) and / or (B) as described below. Preferred examples of the mismatch endonuclease mutant of the present invention include mismatch endonuclease mutants consisting of the polypeptide shown in (A) and / or (B) as described below.

[0044] (A) A polypeptide selected from the group consisting of the following (1) to (4) and having an activity of cleaving a nucleic acid chain having guanine (G) that forms a mismatched base pair in a double-stranded nucleic acid: (1) A polypeptide comprising an amino acid sequence comprising, in the amino acid sequence shown in SEQ ID NO: 1, substitution of serine at position 47 with a basic amino acid residue and substitution of asparagine at position 76 with an acidic amino acid residue; (2) A polypeptide comprising an amino acid sequence comprising a mutation of an additional 1 to 10 amino acid residues in the substituted amino acid sequence described in (1), wherein the mutation is a substitution, deletion, insertion and / or addition, and the amino acid substitution in the substituted amino acid sequence described in (1) is retained; (3) a polypeptide comprising an amino acid sequence having 50% or more homology with the substituted or mutated amino acid sequence described in (1) or (2), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (1) or (2) is retained; and (4) A polypeptide comprising an amino acid sequence having 50% or higher identity with the substituted or mutated amino acid sequence described in (1) or (2), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (1) or (2) is retained.

[0045] (B) A polypeptide selected from the group consisting of the following (5) to (8) and having an activity of cleaving a nucleic acid chain having thymine (T) that forms a mismatched base pair in a double-stranded nucleic acid: (5) A polypeptide comprising an amino acid sequence comprising, in the amino acid sequence shown in SEQ ID NO: 1, substitution of glutamine at position 78 by a basic amino acid residue and substitution of leucine at position 123 by a neutral (polar, uncharged) amino acid residue or a sulfur-containing amino acid residue, or substitution of leucine at position 125 by a hydrophobic amino acid residue or a sulfur-containing amino acid residue; (6) A polypeptide comprising an amino acid sequence comprising a mutation of an additional 1 to 10 amino acid residues in the substituted amino acid sequence described in (5), wherein the mutation is a substitution, deletion, insertion and / or addition, and the amino acid substitution in the substituted amino acid sequence described in (5) is retained; (7) A polypeptide comprising an amino acid sequence having 50% or more homology with the substituted or mutated amino acid sequence described in (5) or (6), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (5) or (6) is retained; and (8) A polypeptide comprising an amino acid sequence having 50% or higher identity with the substituted or mutated amino acid sequence described in (5) or (6), wherein the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (5) or (6) is retained.

[0046] The present invention provides polypeptides having GG-specific mismatch endonuclease activity. Examples of polypeptides having GG-specific mismatch endonuclease activity include, but are not limited to, the polypeptides shown in (A) as described above. Preferred examples of polypeptides having GG-specific mismatch endonuclease activity include, but are not limited to, polypeptides comprising an amino acid sequence contained in the amino acid sequence shown in SEQ ID NO:1, a substitution of serine at position 47 by a basic amino acid residue and a substitution of asparagine at position 76 by an acidic amino acid residue, as described in (A)(1) above. Further examples of polypeptides having GG-specific mismatch endonuclease activity include polypeptides comprising an amino acid sequence contained in SEQ ID NO:1, a substitution of leucine at position 123 by an amino acid having an aliphatic side chain, and the above-mentioned amino acid substitutions at positions 47 and 76. Further preferred examples of polypeptides having GG-specific mismatch endonuclease activity include polypeptides comprising an amino acid sequence comprising, in the amino acid sequence of SEQ ID NO: 1, substitution of serine at position 47 by lysine, arginine or histidine and substitution of asparagine at position 76 by aspartic acid or glutamic acid. Further preferred examples of polypeptides having GG-specific mismatch endonuclease activity also include polypeptides comprising an amino acid sequence comprising, in SEQ ID NO: 1, substitution of leucine at position 123 by alanine, glycine, valine, leucine, isoleucine or proline, and the above-mentioned amino acid substitutions at positions 47 and 76.

[0047] Further, the present invention provides a polypeptide having TT-specific mismatch endonuclease activity. Examples of polypeptides having TT-specific mismatch endonuclease activity include, but are not limited to, the polypeptides shown in (B) as described above. Preferred examples of polypeptides having TT-specific mismatch endonuclease activity include, but are not limited to, a polypeptide comprising an amino acid sequence comprising, in the amino acid sequence shown in SEQ ID NO: 1, a substitution of glutamine at position 78 by a basic amino acid residue, and a substitution of leucine at position 123 by a neutral (polar, uncharged) amino acid residue or a sulfur-containing amino acid residue, or a substitution of leucine at position 125 by a hydrophobic amino acid residue or a sulfur-containing amino acid residue, as described in (B)(5) above. Further preferred examples of the polypeptide having TT-specific mismatch endonuclease activity include polypeptides comprising an amino acid sequence comprising, in the amino acid sequence of SEQ ID NO: 1, substitution of glutamine at position 78 by lysine, arginine or histidine, and substitution of leucine at position 123 by threonine, serine, asparagine, glutamine, tyrosine, cysteine ​​or methionine, or substitution of leucine at position 125 by alanine, glycine, valine, isoleucine, proline, tryptophan, phenylalanine, cysteine ​​or methionine. In a more preferred example of the polypeptide having TT-specific mismatch endonuclease activity, leucine at position 125 may be replaced with an amino acid having an aliphatic side chain other than leucine.

[0048] The polypeptide with GG-specific mismatch endonuclease activity and the polypeptide with TT-specific mismatch endonuclease activity can be in the form of homodimers. When the polypeptide with GG-specific mismatch endonuclease activity is expressed as a monomer in a host (such as Escherichia coli), the monomer naturally combines to form a dimer (homodimer) and thus exhibits activity. The same applies to polypeptides with TT-specific mismatch endonuclease activity.

[0049] In addition, the present invention also provides a polypeptide with GT / TG-specific mismatch endonuclease activity. The example of the polypeptide with GT / TG-specific mismatch endonuclease activity includes but is not limited to the heterodimeric protein comprising the polypeptide shown in (A) and the polypeptide shown in (B) above. The preferred example of the polypeptide with GT / TG-specific mismatch endonuclease activity includes but is not limited to the heterodimeric protein comprising a polypeptide with GG-specific mismatch endonuclease activity and a polypeptide with TT-specific mismatch endonuclease activity. Heterodimeric protein can be produced by expressing a polypeptide with GG-specific mismatch endonuclease activity and a polypeptide with TT-specific mismatch endonuclease activity in a host (such as Escherichia coli, etc.), thereby forming a dimer (heterodimer). However, the method for producing heterodimeric protein is not limited. After expressing the polypeptide with GG-specific mispairing endonuclease activity and the polypeptide with TT-specific mispairing endonuclease activity separately, and dissociating into monomers by any method subsequently, the monomers of the two polypeptides can be mixed to allow recombination. In the preferred method of producing heterodimeric protein, the polypeptide with GG-specific mispairing endonuclease activity and the polypeptide with TT-specific mispairing endonuclease activity are expressed as single polypeptides via joints. The arrangement of the polypeptide with GG-specific mispairing endonuclease activity and the polypeptide with TT-specific mispairing endonuclease activity in a single polypeptide is not limited. They can be arranged in the following order: [polypeptide with GG-specific mispairing endonuclease activity]-[joint]-[polypeptide with TT-specific mispairing endonuclease activity], or [polypeptide with TT specific mispairing endonuclease activity]-[sequential arrangement of joints]-[polypeptide with GG-specific mispairing endonuclease activity]. The joint is preferably a peptide joint. The amino acid sequence and the residue number (length) of the peptide linker are not restricted, as long as the linker does not inhibit heterodimerization and the cutting of double-stranded nucleic acid. In addition, there can be a recognition sequence for the protease used to cut the fusion polypeptide between the polypeptide with mismatched endonuclease activity and the linker, for example Factor Xa, PreScission protease, thrombin, enterokinase, TEV protease (tobacco etch virus protease) etc.

[0050] In addition to the above-mentioned amino acid substitutions described in (A)(1) and (B)(5), the mismatch endonuclease mutants of the present invention may include mutations at other amino acid positions, as long as the mismatch endonuclease mutants do not lose substrate specificity (specificity for GG, TT or GT / TG mismatches). Examples of mutations at other amino acid positions include, but are not limited to, mutations that increase thermotolerance, mutations that increase resistance to inhibitors, and mutations that improve the properties of the mismatch endonuclease, as long as the substrate specificity of the mismatch endonuclease mutants of the present invention is maintained. As used herein, a "mutation" can be any substitution, insertion, deletion or addition of an amino acid.

[0051] For example, the mismatch endonuclease mutant of the present invention including mutations at other amino acid positions may comprise an amino acid sequence comprising mutations of additional 1 to 10 amino acid residues in addition to the above-mentioned amino acid substitutions in (A)(1) and (B)(5).

[0052] In addition, for example, the mismatch endonuclease mutant of the present invention comprising mutations at other amino acid positions may comprise an amino acid sequence having 50% or higher homology with the amino acid sequence of SEQ ID NO: 1 comprising the above-mentioned amino acid substitutions in (A)(1) and (B)(5) or with the amino acid sequence of SEQ ID NO: 1 comprising the above-mentioned amino acid substitutions in (A)(1) and (B)(5) and mutations of 1 to 10 amino acid residues. The 50% or higher homology includes, for example, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, or 75% or higher, preferably 80% or higher, more preferably 85% or higher, further more preferably 90% or higher, and even more preferably 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher amino acid sequence homology.

[0053] In addition, for example, the mismatch endonuclease mutant of the present invention comprising mutations at other amino acid positions may comprise an amino acid sequence having 50% or higher identity with the amino acid sequence of SEQ ID NO: 1 comprising the above-mentioned amino acid substitutions in (A)(1) and (B)(5) or with the amino acid sequence of SEQ ID NO: 1 comprising the above-mentioned amino acid substitutions in (A)(1) and (B)(5) and mutations of additional 1 to 10 amino acid residues. The 50% or higher identity includes, for example, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, or 75% or higher, preferably 80% or higher, more preferably 85% or higher, further preferably 90% or higher, and even more preferably 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher amino acid sequence identity.

[0054] In addition, the present invention includes mismatched endonuclease mutants, which include an affinity tag added to the N-terminus or C-terminus for the purpose of promoting purification. The affinity tag can be a known affinity tag, and its examples include histidine (His) tags, Flag tags, HA tags, c-myc tags, glutathione S-transferase (GST) tags, maltose binding protein (MBP) tags and Strep (II) tags composed of 8 amino acid residues (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) composed of 4 to 8 continuous His residues. The tag can be optionally connected to the mutant of the present invention via a joint comprising 1 to 15 amino acids. In addition, there can be a recognition sequence for the protease for fusion polypeptide cutting between the polypeptide with mismatched endonuclease activity and the joint. The example of the protease includes factor Xa, PreScission protease, thrombin, enterokinase and TEV protease (tobacco etch virus protease). Therefore, such polypeptides comprising affinity tags, linkers and / or recognition sequences for proteases used for fusion polypeptide cleavage are also examples of the mismatched endonuclease mutants of the present invention comprising mutations at other amino acid positions. For example, within the range of 1 to 10 mutant amino acid residue numbers, amino acid homology or amino acid identity as described above, the mismatched endonuclease mutants of the present invention comprising mutations at other amino acid positions may comprise affinity tags, linkers and / or recognition sequences for proteases used for fusion polypeptide cleavage.

[0055] The mismatched endonuclease mutant of the present invention can include artificial amino acids (also referred to as non-natural amino acids). Examples of artificial amino acids include halogenated (chloro, bromo, iodo) tyrosine, tyrosine sulfate, azidotyrosine, acetyl lysine, azidophenylalanine and fluorophenylalanine. The method for replacing natural amino acids with artificial amino acids is not particularly limited, and known methods can be used.

[0056] Examples of the mismatch endonuclease mutants of the present invention include, but are not limited to, mutants consisting of any of the amino acid sequences shown in SEQ ID NOs: 6-15.

[0057] Such mismatch endonuclease mutants of the present invention are suitable for various uses as described below, for example, for use in methods for excluding DNA containing a specific DNA sequence and amplifying and detecting other DNAs.

[0058] The activity of mismatch endonuclease can be measured using a double-stranded nucleic acid containing mispairing as a substrate. Specifically, by reacting the mismatch endonuclease with an excessive amount of double-stranded nucleic acid containing mispairing, and then measuring the amount of nucleic acid cut per unit time to measure the activity. Double-stranded nucleic acids and uncut nucleic acids can be quantitatively cut respectively by electrophoresis, etc. Double-stranded nucleic acids can be double-labeled with fluorescent substances and quenchers so that the increase in fluorescence intensity is detected only when cut. By using double-labeled double-stranded nucleic acids, the fluorescence intensity in the reaction solution is determined at appropriate time intervals to promote activity measurement. It is also possible to study the cutting activity of targeting specific mispairing by changing the mismatched base pairs in the double-stranded nucleic acid as a substrate.

[0059] 2. Nucleic acid encoding the mismatch endonuclease mutant of the present invention The present invention provides a nucleic acid encoding a mismatch endonuclease mutant. Specifically, a nucleic acid encoding the mismatch endonuclease mutant of the present invention is provided.

[0060] Examples of nucleic acids encoding the mismatch endonuclease mutants of the present invention include, but are not limited to, nucleic acids comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NOs: 6 to 15. Preferred examples of nucleic acids encoding the mismatch endonuclease mutants of the present invention include nucleic acids comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NOs: 21 to 30.

[0061] As examples of the mismatch endonuclease mutants of the present invention, the amino acid sequences of the polypeptides prepared in the Examples and their encoding nucleic acid sequences are shown in Table 1, but the present invention is not limited thereto.

[0062] [Table 1]

[0063] The nucleic acid of encoding mispairing endonuclease mutant of the present invention is not particularly limited, as long as it is composed of the codons of the protein that can be expressed in the employed host and has reverse transcriptase activity. Can carry out codon optimization to nucleic acid, to allow to express or increase expression level in the host. Codon optimization is preferably carried out by the method commonly used in this area.

[0064] 3. Expression vector containing a nucleic acid encoding the mismatch endonuclease mutant of the present invention The expression vector of the present invention contains a nucleic acid encoding the mismatch endonuclease mutant of the present invention, and an expression control sequence operably linked to the nucleic acid.

[0065] The expression vector used in the present invention can be an expression vector commonly used in the art, and is not particularly limited. A vector that can replicate autonomously in a host cell, or a vector that can be integrated into a host chromosome can be used. A vector compatible with the host can be used.

[0066] The example of the expression vector of the nucleic acid of inserting the mismatched endonuclease mutant of the present invention comprises plasmid vector, phage vector and viral vector.Plasmid vector can be the plasmid that is suitable for the host to be used, and the example of such plasmid comprises the plasmid derived from Escherichia coli, the plasmid derived from the bacterium belonging to Bacillus, and the plasmid derived from yeast.Such plasmid vector is well known to those skilled in the art, and there are many commercially available plasmid vectors.These known plasmids and the plasmids changed thereof can be used in the present invention.Phage vector can be such as lambda phage etc.Viral vector can be such as animal virus, such as retrovirus or vaccinia virus, or insect virus, such as baculovirus.In addition, many heterologous protein expression systems using yeast, insect cell and mammalian cell as host have been constructed, and can be commercially available.Such expression system can be used for producing the mismatched endonuclease mutant of the present invention.

[0067] The promoter to be included in the expression vector of the present invention can be selected according to the host. For example, for E. coli, a promoter derived from E. coli or a bacteriophage or a modified promoter thereof can be used, including but not limited to the trp promoter, the lac promoter, the PL promoter, and the PR promoter. Further, an expression system containing a combined promoter derived from a bacteriophage and an RNA polymerase gene derived from a bacteriophage (e.g., a pET expression system, etc.) can be used.

[0068] In order to facilitate the purification of the expressed polypeptide, the expression vector of the present invention may further comprise a nucleic acid encoding an affinity tag. The nucleic acid encoding the affinity tag is inserted into the vector, so that the fusion protein of the mismatched endonuclease mutant of the present invention and the affinity tag can be expressed. Examples of affinity tags include, but are not limited to, histidine (His) tags, glutathione S-transferase (GST) tags, maltose binding protein (MBP) tags, and Strep (II) tags consisting of 8 amino acid residues (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). The tag can be added to the 5' end side and / or 3' end side of the nucleic acid encoding the mismatched endonuclease mutant of the present invention. The tag can be appropriately added to a position that does not interfere with expression and tag function. The tag is preferably a tag that can be cut during or after the purification of the expressed polypeptide. Examples of such cleavable tags include, but are not limited to, tags comprising a nucleic acid encoding a recognition sequence for a protease for cutting a fusion polypeptide, such as Factor Xa, PreScission protease, thrombin, enterokinase, and TEV protease (tobacco etch virus protease).

[0069] The expression vector of the present invention also contains one or more expression control sequences. Examples of expression control sequences include, but are not limited to, promoters, genes involved in regulating promoters, ribosome binding sequences, polyadenylation signals, transcription termination sequences (transcription terminators) and enhancers. The expression vector of the present invention may further include genes encoding replication origins or markers (drug resistance genes, fluorescent markers or luminescent markers) for selecting transformants, and nucleotide sequences for enhancing translation efficiency.

[0070] 4. Cells transformed with the expression vector of the present invention The cell (host) transformed with the expression vector expressing the mismatched endonuclease mutant of the present invention can be a host commonly used in the art, and is not particularly limited. Its examples include bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, filamentous fungi, insect cells, eukaryotic cells and animal cells (mammalian cells, including human cells, etc.).

[0071] When using prokaryotic cells as host cells, examples of the host cells include bacteria belonging to the genus Escherichia, such as Escherichia coli; Bacillus, such as Bacillus subtilis; Pseudomonas, such as Pseudomonas putida, and Rhizobium, such as Rhizobium meliloti. Escherichia coli that can be used to produce heterologous proteins are well known to those skilled in the art, and there are many commercially available strains of Escherichia coli (e.g., Escherichia coli BL21T1R, Escherichia coli BL21, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli JM109, Escherichia coli HB101, etc.). Further, Bacillus subtilis MI114, Bacillus subtilis 207-21, etc. belonging to the genus Bacillus, Brevibacillus choshinensis, etc. belonging to the genus Brevibacillus, etc. are known as hosts for producing heterologous proteins. The above host cells and suitable expression vectors can be used in combination to produce the mismatch endonuclease mutants of the present invention. Preferably, Escherichia coli BL21T1R or BL21DE3, which are strains of Escherichia coli BL21, can be used.

[0072] The method for introducing the expression vector into the host is not particularly limited, as long as it can introduce nucleic acid into the host. Examples of the method include methods using calcium ions, electroporation, spheroplasting, and lithium acetate methods. The method for introducing the expression vector into insect cells is not particularly limited, as long as it can introduce DNA into insect cells, and examples thereof include calcium phosphate method, liposome transfection, and electroporation. When using a phage vector or a viral vector, the host cell can be infected by a method suitable for the vector, and a transformant expressing the mismatched endonuclease mutant of the present invention can be obtained thereby.

[0073] Transformants are cultured. The mismatched endonuclease mutants of the present invention can be collected from the culture of the transformants. The culture conditions are not particularly limited as long as they are suitable for the expression vector used, the host used, etc. For example, Escherichia coli BL21DE3 is transformed using a pET vector, and the transformant is inoculated into an LB medium and cultured at 37°C with shaking. When the OD value of the culture reaches 0.2-0.8, IPTG is added to the culture medium, and then the shaking culture is continued to induce the expression of the desired protein, for example, at 15-30°C for 2-5 hours, preferably at 25°C for 4-5 hours. Then, the culture is centrifuged, and the bacterial cells thus obtained are washed, and then ultrasonically treated or lysed with lysozyme to obtain a total cell extract containing the mutants of the present invention. Since the extract contains a large amount of impurities, the mutants of the present invention are preferably purified by a suitable combination of purification methods used in the art, such as ammonium sulfate precipitation, anion exchange columns, cation exchange columns, gel filtration columns, affinity chromatography columns, dialysis, etc. By using an affinity carrier suitable for the characteristics of the affinity tag, the mutant with the affinity tag can be easily purified. Furthermore, in addition to IPTG, other necessary inducers, such as L-arabinose, may be added at an appropriate time depending on the type of host or expression vector used.

[0074] 5. Method for producing nucleic acids encoding the mismatch endonuclease mutants of the present invention The method for producing nucleic acid of the present invention includes, for example, in the nucleic acid encoding mispairing endonuclease, the codon encoding the serine at the position of the 47th position in the amino acid sequence corresponding to SEQ ID NO:1 is replaced with the step of the codon encoding basic amino acids, the mispairing endonuclease polypeptide PF_RS00065 or its mutant polypeptide W77F, or the homologue of RS_00065. The codon of serine is preferably replaced with the codon of arginine or lysine, more preferably replaced with the codon of arginine. Similarly, the codon of the amino acid residue in other positions can also be replaced.

[0075] As described above in Section 1, the mismatch endonuclease mutant of the present invention may comprise mutations at other amino acid positions. Even in this case, codons may be similarly substituted.

[0076] Preferred examples of the nucleic acid encoding the mismatch endonuclease mutant comprising the above-mentioned codon substitution include the nucleic acids of SEQ ID NOs: 16-30.

[0077] Codon replacement can be carried out by known methods, including but not limited to introducing mutation by known methods, for example, site-specific mutagenesis using primers for introducing mutation, and artificial synthesis of nucleic acids with mutant sequence (or a part of sequence). Further, in order to allow expression or increase the purpose of expression level in the host used, codon optimization can be carried out. Codon optimization can be carried out by methods commonly used in this area.

[0078] The method of producing the nucleic acid of the present invention may further comprise replacing codons in the nucleic acid encoding the mismatch endonuclease mutant for the purpose of stabilizing and increasing protein production in the host.

[0079] For the method of producing the nucleic acid of the present invention, the nucleic acid described in detail in Section 2 above can be applied.

[0080] 6. Double-stranded nucleic acid cleavage method of the present invention The method for cleaving a double-stranded nucleic acid of the present invention is carried out by treating a double-stranded nucleic acid having a mismatch in an appropriate buffer containing a divalent metal ion (e.g., a magnesium ion) using the mismatch endonuclease mutant of the present invention described in Section 1 above. According to the method of the present invention, a double-stranded nucleic acid containing a GG mismatch, a TT mismatch, a GT mismatch, or a TG mismatch can be cleaved.

[0081] For the cutting method of double-stranded nucleic acid of the present invention, the double-stranded nucleic acid with mispairing can be a double-stranded nucleic acid containing mispairing inside (between two base pairs of normal pairing). The double-stranded nucleic acid with mispairing can not only be a double-stranded nucleic acid containing one mispairing, but also a double-stranded nucleic acid containing two or more mispairings at intervals, or a double-stranded nucleic acid containing two or more continuous mispairings. For the cutting method of double-stranded nucleic acid of the present invention, the example of mispairing includes 1-8 continuous mispairings preferably present in the double-stranded nucleic acid, more preferably 1-4 continuous mispairings, further preferably 2 continuous mispairings, and 1 mispairing. In the cutting method of double-stranded nucleic acid of the present invention, when there are two or more mispairings in the double-stranded nucleic acid, the two or more mispairings can be mispairings of the same kind, or can be mispairings of different kinds.

[0082] In addition, oligodeoxynucleotides as disclosed in WO2014 / 142261 etc. can be used. As used herein, the oligodeoxynucleotide is an oligodeoxynucleotide designed to produce one or more mismatches when hybridizing with a nucleic acid having a specific nucleotide sequence (e.g., having a gene mutation of interest). For use as a probe for mutation detection, the oligodeoxynucleotide can be labeled with a fluorescent substance and a quenching substance at both ends. The length of the oligodeoxynucleotide can be appropriately determined so that the oligodeoxynucleotide can hybridize with a nucleic acid having a specific nucleotide sequence under the reaction conditions to be performed. When the oligodeoxynucleotide is hybridized with a nucleic acid having a specific nucleotide sequence, the mismatch is preferably produced at a position of at least 3 nucleotides from the 5' end and the 3' end of the oligodeoxynucleotide.

[0083] In addition, when the gene mutation to be detected does not match the substrate specificity of the mismatched endonuclease and the gene mutation cannot be analyzed, the inhibitory oligonucleotide disclosed in WO2016 / 152812 etc. can be used. As used herein, an inhibitory oligonucleotide is an oligonucleotide that forms at least one mismatch when an oligonucleotide is hybridized with a non-target nucleic acid, and forms more mismatches when an oligonucleotide is hybridized with a target nucleic acid than when an oligonucleotide is hybridized with a non-target nucleic acid. The number of at least one mismatch formed when an inhibitory oligonucleotide is hybridized with a non-target nucleic acid is not particularly limited, as long as selective cutting of non-target nucleic acids occurs, and depending on the length of the inhibitory oligonucleotide, its examples include 1-7, 1-5 or 1-3 mismatches. When the number of at least one mismatch is expressed as a percentage of the length of the inhibitory oligonucleotide, for example, at least one mismatch accounts for 1-20%, 3-15% or 4-8% of the length of the inhibitory oligonucleotide. As an example of this aspect, a combination of a target nucleic acid and a non-target nucleic acid whose nucleotide sequence differs only by one base is described. When the inhibitory oligonucleotide hybridizes to the target nucleic acid, a mismatch is formed between the bases that are different between the inhibitory oligonucleotide and the target nucleic acid / non-target nucleic acid (hereinafter sometimes referred to as the first mismatch), and another mismatch is formed (hereinafter sometimes referred to as the second mismatch).

[0084] The first mismatched base pair involves bases that differ between the target nucleic acid and the non-target nucleic acid, regardless of whether the mismatched base pair is recognized and cleaved by a coexisting polypeptide having mismatch endonuclease activity.

[0085] The second mismatch is a mismatch base pair that is recognized and cleaved by a coexisting polypeptide with mismatch endonuclease activity. For example, when a polypeptide with mismatch endonuclease activity is used to recognize and cleave guanine base-guanine base, guanine base-thymine base, or thymine base-thymine base, the inhibitory oligonucleotide is designed to form a mismatch selected from the above mismatch base pairs.

[0086] When the inhibitory oligonucleotide hybridizes to a non-target nucleic acid, a second mismatch is formed without forming a first mismatch.

[0087] Design and use inhibitory oligonucleotides with the above-mentioned characteristics, so that non-target nucleic acids can be selectively cut by polypeptides with mismatched endonuclease activity. The present invention is not limited to the use of inhibitory oligonucleotides that can form one or two mispairings as described above. Can design and use inhibitory oligonucleotides that can form 3 or more mispairings, as long as the selective cutting of required nucleic acid occurs. In this case, at least one mispairing formed in target nucleic acid and non-target nucleic acid can be the second mispairing, and other mispairings may or may not be recognized and cut by polypeptides with mismatched endonuclease activity. Described 3 or more mispairings are preferably recognized and cut by polypeptides with mismatched endonuclease activity.

[0088] The oligodeoxynucleotides disclosed in WO2014 / 142261 and the inhibitory oligonucleotides disclosed in WO2016 / 152812 and the like are composed of DNA, or may partially contain nucleotide analogs or RNA. In other words, the oligodeoxynucleotides and the inhibitory oligonucleotides are not particularly limited as long as they have a structure that forms a double-stranded nucleic acid having at least one mismatch when hybridized with a non-target nucleic acid, and the mismatch is recognized and cleaved by a coexisting polypeptide having mismatch endonuclease activity.

[0089] The example of double-stranded nucleic acid with mispairing cut by the method of the present invention includes PCR product, nucleic acid derived from biological sample, such as genomic DNA or its fragment, and synthetic nucleic acid. Double-stranded nucleic acid with mispairing can be a mixture of nucleic acid from biological sample, or a mixture of melting-reannealing of nucleic acid from biological sample and synthetic nucleic acid. For example, when the nucleic acid containing mutation and wild-type nucleic acid are mixed, melted and reannealed, mispairing is formed and the cutting of mispairing endonuclease occurs at the position of mispairing. The size of the nucleic acid fragment obtained after cutting by mispairing endonuclease can be observed to assess the presence or absence of mutation and the position of mutation. Using mispairing endonuclease of the present invention, it is allowed to add mutation analysis of the reaction solution for nucleic acid amplification (such as PCR) by simply adding this mispairing endonuclease. For PCR, it is known that when the number of cycles is increased to higher than a certain number, the increase of amplification effect is not found. This is mainly because the primer or substrate dNTP added is exhausted or the competition annealing between primer and reaction product. At this time, annealing occurs between reaction products. If there are both a template with a mutation and a wild-type template, a mismatch is formed at the mutation position by annealing between the reaction product amplified from the mutant template and the reaction product amplified from the wild-type template. Thus, mutation analysis is made possible by simply performing PCR for a greater number of cycles than usual in the presence of the mismatch endonuclease of the present invention. In other words, the present invention provides a method for analyzing mutations, the method comprising treating a double-stranded nucleic acid with the mismatch endonuclease described in Section 1 above.

[0090] The cleavage method of double-stranded nucleic acid of the present invention can be carried out in the presence of acidic macromolecular substances. It has been found that acidic macromolecular substances have the effect of controlling the mismatch recognition and cleavage activity of polypeptides by the coexistence of acidic macromolecular substances. Acidic macromolecular substances play a greater role in samples containing a small amount of nucleic acid. Preferred examples of acidic macromolecular substances include polyanions. Preferred examples of acidic macromolecular substances also include acidic polysaccharides with sugar backbones and acidic polysaccharides with linear carbon chains. As acidic macromolecular substances, one or more substances selected from the following can be used: polysaccharides containing sulfated fucose, dextran sulfate, carrageenan, heparin, rhamnosyl sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, hyaluronic acid, alginic acid, pectin, polyglutamic acid, polyacrylic acid, polyvinyl sulfate, polystyrene sulfate, and salts thereof, and nucleic acids different from target nucleic acids and non-target nucleic acids.

[0091] The double-stranded nucleic acid cleavage method of the present invention can also be performed in combination with the use of proliferating cell nuclear antigen (PCNA).

[0092] 7. Double-stranded nucleic acid amplification method of the present invention The cutting method of double-stranded nucleic acid of the present invention can even be carried out during the process of nucleic acid amplification reaction. By adding mismatched nuclease to the reaction mixture for nucleic acid amplification, double-stranded nucleic acid with mismatch is cut, and the mismatch is formed by incorporating wrong nucleotides during the amplification process. Thus, the amplification of nucleic acids with sequences different from the template nucleic acid before the reaction starts is suppressed. Therefore, nucleic acid amplification with a reduced error rate is made possible.

[0093] That is, the present invention provides a method for nucleic acid amplification, which includes the step of cutting a double-stranded nucleic acid with a mismatch by using the mismatch endonuclease mutant of the present invention described in Part 1 above. The step of cutting a double-stranded nucleic acid with a mismatch can be performed simultaneously with the nucleic acid amplification step. In addition, as one aspect of the present invention, a composition is provided, which comprises (a) a DNA polymerase, (b) at least one pair of oligonucleotide primers, and (c) the mismatch endonuclease described in Part 1 above.

[0094] Similarly, as one aspect of the present invention, a nucleic acid amplification method is also provided, which includes the steps of preparing the following composition, wherein the composition contains the above-mentioned composition for nucleic acid amplification reaction and a nucleic acid molecule as a template; and the step of performing nucleic acid amplification by reacting the obtained composition under appropriate conditions.

[0095] The above composition may further include at least one selected from the following: reaction buffer, divalent metal ions, deoxyribonucleotides, oligonucleotide probes and intercalating dyes. When the composition is used for nucleic acid amplification reaction, the composition may further include nucleic acids as templates for nucleic acid amplification reaction. In the composition, deoxyribonucleotides may contain nucleotide analogs. Reaction buffer refers to a compound or mixture having the ability to moderate changes in hydrogen ion concentration (pH) in a reaction solution. Since a mixed solution of a weak acid and its salt, or a weak base and its salt generally has a strong buffering effect, the mixed solution is widely used as a reaction buffer for the purpose of controlling pH. Examples of reaction buffers used in the present invention include Good's buffers, such as Tris-HCl and HEPES-KOH, and phosphate buffers, such as sodium phosphate buffers. Examples of divalent metal ions include magnesium ions, manganese ions, zinc ions and cobalt ions. Divalent metal ions can be provided as salts, such as chlorides, sulfates or acetates.

[0096] An example of the method for amplifying the double-stranded nucleic acid of the present invention is a method for amplifying DNA, but the present invention is not particularly limited thereto. Examples of the method for amplifying DNA include polymerase chain reaction (PCR), MDA (multiple displacement amplification), and isothermal nucleic acid amplification such as ICAN and LAMP.

[0097] The concentration of the polypeptide having mismatch endonuclease activity in the above composition for nucleic acid amplification reaction can be determined by measuring the concentration that does not inhibit the DNA amplification reaction or the concentration effective for cleavage of double-stranded nucleic acid having mismatches in each reaction system, as appropriate.

[0098] As at least one pair of primers of the above-mentioned composition for nucleic acid amplification reaction, two or more primers suitable for each nucleic acid amplification method are selected. The primers can be DNA primers, RNA primers or chimeric primers in which a part of the DNA molecule is replaced by RNA, as long as the desired amplification is obtained. The primers can also be primers containing known nucleic acid analogs, and labeled primers, for example, with fluorescent dyes for detection purposes.

[0099] In addition, as another aspect of the composition of the present invention, the composition may include a component for preventing carryover. Examples of such a component include dUTP and uracil N-glycosidase.

[0100] The amplification method of double-stranded nucleic acid of the present invention can be carried out in the presence of the above-mentioned acidic macromolecular substance. The acidic macromolecular substance plays a greater role in samples containing a small amount of nucleic acid. Preferred examples of acidic macromolecular substances are described above for the method for selectively cutting non-target nucleic acids of the present invention. The method of the present invention can be further carried out in combination with the above-mentioned proliferating cell nuclear antigen (PCNA) or its mutant.

[0101] 8. Method for inhibiting nucleic acid amplification of the present invention The method of inhibiting nucleic acid amplification of the present invention comprises using the mismatch endonuclease mutant of the present invention described in Section 1 above and an appropriately designed oligodeoxynucleotide or inhibitory oligonucleotide to inhibit the amplification of a nucleic acid having a specific nucleotide sequence in a nucleic acid amplification reaction.

[0102] Therefore, one aspect of the present invention provides a method for inhibiting the amplification of a nucleic acid having a specific nucleotide sequence in a nucleic acid amplification reaction, the method comprising the steps of conducting a nucleic acid amplification reaction in the presence of: (a) an oligodeoxynucleotide or inhibitory oligonucleotide designed to produce one or several mismatches when hybridizing with a nucleic acid comprising a specific nucleotide sequence; (b) a DNA polymerase; (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having mismatch endonuclease activity. A further aspect of the present invention provides a method for preferentially amplifying a target nucleic acid, the method comprising inhibiting the amplification of a nucleic acid having a specific nucleotide sequence by using the above method, the specific nucleotide sequence containing one or several nucleic acids different from the nucleotide sequence of the target nucleic acid.

[0103] (a) The oligodeoxynucleotide or inhibitory oligonucleotide is not particularly limited, as long as it is a single-stranded DNA having a nucleotide sequence designed to form one or more mismatches when hybridizing with a nucleic acid comprising a specific nucleotide sequence. The oligodeoxynucleotide or inhibitory oligonucleotide may be a so-called chimeric oligodeoxynucleotide in which a portion of the DNA molecule is replaced by RNA. The 3'-end of the oligodeoxynucleotide or inhibitory oligonucleotide may be modified to inhibit the extension reaction of DNA polymerase to single-stranded DNA, but the present invention is not particularly limited thereto. Examples of modifications include amination. The oligodeoxynucleotide or inhibitory oligonucleotide may be protected from cleavage by deoxyribonucleases by thiophosphorylation or other modifications, as long as the nucleic acid bound to the oligodeoxynucleotide or inhibitory oligonucleotide is subjected to cleavage by a polypeptide having mismatch endonuclease activity. The oligodeoxynucleotide or inhibitory oligonucleotide may be labeled with a fluorescent dye, a quencher, etc. for the purpose of detection.

[0104] The length of the oligodeoxynucleotide or inhibitory oligonucleotide can be appropriately determined so that the oligodeoxynucleotide or inhibitory oligonucleotide can hybridize with a nucleic acid having a specific nucleotide sequence under the reaction conditions performed. The position of the mismatch generated when the oligodeoxynucleotide or inhibitory oligonucleotide hybridizes with a nucleic acid having a specific nucleotide sequence is preferably at least 3 nucleotides from both the 5' end and the 3' end of the oligodeoxynucleotide or inhibitory oligonucleotide.

[0105] For example, when performing the method of the present invention to inhibit the amplification of a nucleic acid having a specific base sequence by a method including a reaction at a high temperature (such as PCR), it is preferred to use a thermostable mismatch endonuclease. The mismatch endonuclease described in Section 1 above includes a thermostable enzyme that is not inactivated in PCR, and such enzymes are suitable for the above-mentioned purposes.

[0106] Therefore, the present invention also provides a composition for nucleic acid amplification reaction, comprising the following (a) to (d): (a) an oligodeoxynucleotide designed to produce one or several mismatches when hybridizing to a nucleic acid comprising a specific nucleotide sequence, or an inhibitory oligonucleotide or its complementary strand; (b) DNA polymerase; (c) at least one pair of oligonucleotide primers; and (d) The mismatch endonuclease mutant of the present invention as described in Section 1 above.

[0107] The composition may further comprise at least one selected from the group consisting of a reaction buffer, a divalent metal ion, a deoxyribonucleotide, an oligonucleotide probe, and an intercalating dye. The composition may further comprise a nucleic acid as a template for a nucleic acid amplification reaction. The composition may further comprise dUTP or uracil N-glycosidase.

[0108] The method for inhibiting the amplification of a nucleic acid having a specific nucleotide sequence in a nucleic acid amplification reaction of the present invention can be carried out by any nucleic acid amplification method. Preferred examples of such nucleic acid amplification methods are, but are not limited to, methods for amplifying DNA. Examples of methods for amplifying DNA include PCR, MDA, and isothermal nucleic acid amplification, such as ICAN and LAMP.

[0109] The method for suppressing the amplification of nucleic acid with specific nucleotide sequence in nucleic acid amplification reaction of the present invention can be applied to any nucleic acid. When the method is applied to DNA amplification, such DNA can be DNA present in artificially prepared DNA mixture, sample derived from environment, biological sample or DNA in DNA mixture prepared by the above-mentioned sample. Examples of biological samples include but are not limited to samples derived from mammals (such as people). Examples of DNA mixtures include but are not limited to mixtures of fragments of genomic DNA, mixtures of cDNA produced from mRNA by reverse transcription reaction, and mixtures of multiple PCR products. Examples of DNA with specific nucleotide sequence suppressed for its amplification include reverse transcription products from rRNA that are still not separated, and small molecule DNA produced by primer pairing. In the case of amplified gene library followed by functional screening, a library capable of more effectively retrieving unknown genes can be produced by suppressing the amplification of DNA with positive known gene sequences.

[0110] In the method of the present invention, the concentration of the polypeptide having mismatch endonuclease activity can be determined by measuring the concentration that does not inhibit the DNA amplification reaction in each reaction system, or the concentration that is effective for the cleavage of double-stranded nucleic acids with mismatches, as appropriate. The concentration of the oligonucleotide or inhibitory oligodeoxynucleotide can be determined by optimizing the concentration used by considering the template amount and the amplification efficiency of the target DNA. For example, the concentration of the oligodeoxynucleotide or inhibitory oligonucleotide can be 0.1 to 10 times the concentration of the primer used for the amplification reaction.

[0111] As described above, the present invention provides a method for preferentially amplifying a target nucleic acid. The method may further include a step of detecting the amplified target DNA. Hereinafter, this aspect of the present invention may be referred to as "the method for detecting nucleic acid of the present invention". For example, according to the detection method of the present invention, wherein DNA is used as a target to be detected, even when a DNA that is not a target to be detected (DNA with a specific nucleotide sequence) is excessively present in large quantities relative to a DNA (target DNA) as a target to be detected, by means of an oligonucleotide or inhibitory oligonucleotide in a method of the present invention for inhibiting amplification of a nucleic acid with a specific nucleotide sequence in a nucleic acid amplification reaction and a polypeptide with mismatched endonuclease activity, amplification with a non-target DNA as a template is suppressed, and the target DNA to be detected can be detected thereby.

[0112] The method for inhibiting nucleic acid amplification of the present invention can be carried out in the presence of the above-mentioned acidic macromolecular substance. The acidic macromolecular substance plays a greater role in samples containing a small amount of nucleic acid. Preferred examples of acidic macromolecular substances are described above for selectively cutting the method for non-target nucleic acid of the present invention. The method of the present invention can be further carried out in combination with the above-mentioned proliferating cell nuclear antigen (PCNA) or its mutant.

[0113] 9. Method for detecting nucleic acid of the present invention The method for detecting nucleic acid of the present invention includes suppressing the amplification of non-target nucleic acid by the method for suppressing nucleic acid amplification of the present invention described in the above-mentioned Section 8, and thus preferably amplifying the step of target nucleic acid, and detecting the step of amplified target nucleic acid. The method for detecting nucleic acid of the present invention can detect the wild type and mutant type of nucleic acid corresponding to the gene having mutation in the known gene, for example, differently. When the detection method of the present invention is carried out using DNA comprising wild-type nucleotide sequence as the nucleic acid (non-target nucleic acid) with specific nucleotide sequence, a small amount of mutant allele can be detected in the case of excessively large amounts of normal alleles (i.e., DNA with wild-type nucleotide sequence) present. For example, the method of the present invention can be used to detect circulating tumor DNA, or to detect a small amount of fetal DNA sequence contained in maternal blood. Examples of mutations include microdeletions and point mutations. The polymorphism produced by point mutations is called single nucleotide polymorphism (SNP). As used herein, DNA with mutant nucleotide sequence in SNP is sometimes referred to as DNA with single nucleotide polymorphism mutation.

[0114] Preferred examples of a small amount of mutant alleles include, but are not limited to, nucleic acids containing at least one single nucleotide polymorphism, selected from single nucleotide polymorphism mutations used as tumor markers, single nucleotide polymorphism mutations associated with the therapeutic effects of agents for cancer treatment, and single nucleotide polymorphism mutations known to be associated with cell carcinogenesis. Examples of SNPs include those often found in tumor cells, and those known to be associated with the therapeutic effects or carcinogenic effects of agents for the treatment of cancer. Examples of such SNPs include SNPs of K-ras genes, B-raf genes, and epidermal growth factor receptor (EGFR) genes. Somatic mutations in the K-ras gene are common in colorectal cancer, lung adenocarcinoma, thyroid cancer, etc. Somatic mutations in the B-raf gene are common in colorectal cancer, malignant melanoma, papillary thyroid cancer, non-small cell lung cancer, lung adenocarcinoma, etc. Somatic mutations in the EGFR gene are common in various solid tumors. It is known that when the EGFR gene in cancer tissue has a specific single nucleotide polymorphism mutation, it is likely to be effective to treat cancer using an EGFR inhibitor, such as gefitinib or erlotinib. On the contrary, it is known that when the K-ras gene in cancer tissue has a single nucleotide polymorphism mutation, cancer may be resistant to EGFR inhibitors.

[0115] The detection method of the present invention can be performed using DNA obtained after treating a composition containing methylated DNA extracted from a sample from an organism with bisulfite as a raw material. According to the detection method of the present invention, a small amount of methylated alleles can be detected in the presence of an excessive amount of non-methylated alleles, or a small amount of non-methylated alleles can be detected in the presence of an excessive amount of methylated alleles.

[0116] As the treatment using bisulfite, a known bisulfite method for detecting methylated DNA can be used. By treatment, non-methylated cytosine becomes uracil, and methylated cytosine does not change. When the reaction solution treated with bisulfite is subjected to PCR amplification, uracil becomes thymine, and methylated cytosine becomes cytosine. In other words, a small amount of methylated alleles are detected in the case of excessively large amounts of non-methylated alleles in a specific site, and a small amount of non-methylated alleles are detected in the case of excessively large amounts of methylated alleles, corresponding to the presence of cytosine in the case of excessively large amounts of thymine, and the presence of thymine in the case of excessively large amounts of cytosine. When the amplification of excessively large amounts of DNA containing thymine or cytosine is suppressed, it is easy to check the presence of a small amount of methylated alleles or non-methylated alleles.

[0117] For the step of detecting target nucleic acid in the detection method of the present invention, electrophoresis, nucleotide sequence analysis or real-time PCR using probes (such as cycle probes or TaqMan probes) can be used. For these detection methods, conventional techniques can be directly used. Specifically, the application of high-resolution melting (HRM) analysis method allows amplification and detection of interested DNA by one step, and thus realizes quick and simple inspection of interested DNA.

[0118] The method for detecting nucleic acid of the present invention can be carried out in the presence of the above-mentioned acidic macromolecular substance. The acidic macromolecular substance plays a greater role in samples containing a small amount of nucleic acid. Preferred examples of acidic macromolecular substances are as described above for the method for selectively cutting non-target nucleic acid of the present invention. The method of the present invention can be further carried out in combination with the above-mentioned proliferating cell nuclear antigen (PCNA) or its mutant. Example Hereinafter, the present invention will be specifically described with reference to Examples, but the scope of the present invention is not limited to these Examples.

[0120] Experimental method 1: (1-1) Preparation of mismatch endonuclease mutants The nucleotide sequence of the gene encoding the polypeptide PF_R00065 from Pyrococcus furiosus (RefSeq ID: WP_11011124.1, SEQ ID NO: 1) is shown in SEQ ID NO: 16. Based on the above nucleotide sequence, a mutation was introduced into a specific position by a conventional method to prepare an artificial gene. The artificial gene thus obtained was introduced into a plasmid pET6xHN-C (manufactured by Takara Bio USA) using an In-Fusion (registered trademark) HD ​​cloning kit (manufactured by TakaraBio USA). The plasmid thus obtained has a nucleotide sequence encoding a mismatch endonuclease mutant, and a histidine tag added to the C-terminal side.

[0121] Then, the plasmid was used to transform the Escherichia coli BL21 DE3 strain (manufactured by Takara Bio Inc.) and cultured overnight at 37°C on a 1.5% agarose LB plate containing 100 μg / ml ampicillin. Three single colonies were selected from the plate, inoculated in an LB medium containing 100 μg / ml ampicillin (hereinafter referred to as LB-AP medium), and cultured overnight at 37°C with shaking. Then, 300 μl of the culture was inoculated into 6 ml of LB-AP medium and cultured overnight at 37°C with shaking. When the OD600 value reached 0.6, IPTG was added to the culture at a final concentration of 1 mM, and then, it was further incubated at 25°C for 4 hours to induce the expression of the target gene. Then, when the OD600 value reached 4, the bacterial cells were collected.

[0122] The bacterial cells thus obtained are suspended in 400 μl of a solution containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol and 0.15% Triton X-100 (hereinafter referred to as buffer S), and ultrasonic wave treatment is performed three times for 30 seconds at 4 ° C using a sonicator (manufactured by Sonic & Materials). Thus, the suspension becomes transparent. After ultrasonic wave treatment, the suspension is centrifuged at 4 ° C with 11000 × g for 10 minutes, and the supernatant is collected. The crude extract thus obtained is subjected to Ni resin purification.

[0123] The purification of Ni resin is carried out as follows. Specifically, 50 μl Ni-NTA agarose (manufactured by Qiagen) is placed in a 1.5 ml tube, washed twice with 250 μl sterile distilled water, and then balanced twice with 250 μl of buffer A (50mM Tris-HCl pH7.5, 300mM NaCl, 5% glycerol and 5mM imidazole). The balanced Ni-NTA agarose is suspended in 400 μl crude extracts, and allowed to stand for 30 minutes. Then, the suspension is centrifuged at 12000rpm for 10 minutes at 4 ℃. After removing the supernatant, the Ni-NTA agarose is washed 3 times with 100 μl buffer A. Then, with 100 μl buffer B (50mM Tris-HCl pH7.5, 300mM NaCl, 5% glycerol and 300mM imidazole), the adsorbate is eluted from the Ni-NTA agarose. The eluate thus obtained was used as a mismatch endonuclease mutant solution in the following experiment.

[0124] (1-2) Preparation of heterodimeric mismatch endonuclease mutants Based on the gene encoding the mutant prepared in (1-1), a gene encoding a heterodimer mismatch endonuclease mutant was prepared. In the same manner as (1-1), an artificial gene was prepared by connecting a gene encoding a GG-specific mismatch endonuclease mutant and a gene encoding a TT-specific mismatch endonuclease mutant in series via a linker (SEQ ID NO: 35).

[0125] (2) Evaluation of substrate specificity of mismatch endonuclease mutants (2-1) Nucleic acid NucS-template-T (SEQ ID NO: 31), NucS-template-G (SEQ ID NO: 32), NucS-probe-T (SEQ ID NO: 33), and NucS-probe-G (SEQ ID NO: 34) were chemically synthesized by a known method as a double-stranded DNA of a nucleic acid-forming substrate. A double-stranded DNA consisting of a combination of a template and a probe was used as a substrate. FAM and Eclipse (registered trademark) dark quencher were added to the 5' end and 3' end of the two probes, respectively.

[0126] (2-2) Cutting Specificity By mixing 2.5 μl of 10x buffer, 1 μl of 25 μM template, 1.5 μl of 5 μM probe, 5 μl of mismatched endonuclease mutant solution and 15 μl of sterile water, a total of 25 μl of mixture is prepared. Using a thermal cycler, a cycle of 55°C for 60 seconds is repeated for 60 cycles. Alternatively, using a thermal cycler, a cycle of 55°C for 15 seconds is repeated for 60 cycles. The composition of the 10x buffer is 500mM Tris-HCl (pH 9.2), 140mM (NH4) 2SO4, 100mM KCl, 25mM MgCl2 and 0.1% BSA. The composition of the buffer for enzyme dilution is 25mM Tris-HCl (pH 8.0), 50mM KCl, 0.1mM DTT, 0.01% gelatin, 0.5% Nonidet P-40, 0.5% Tween-20 and 0.09% BSA.

[0127] Example 1: Preparation of mismatch endonuclease mutants (1) Mismatch endonuclease mutant S47K+N76D According to experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the serine at position 47 and the asparagine at position 76 of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with lysine and aspartic acid, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 6 and SEQ ID NO: 21, respectively.

[0128] (2) Mismatch endonuclease mutant S47R+N76D+L123A According to the experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the serine at position 47, the asparagine at position 76, and the leucine at position 123 of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine, aspartic acid, and alanine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 7 and SEQ ID NO: 22, respectively.

[0129] (3) Mismatched endonuclease mutant S47R+N76D+L123G According to the experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the serine at position 47, the asparagine at position 76, and the leucine at position 123 of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine, aspartic acid, and glycine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 8 and SEQ ID NO: 23, respectively.

[0130] (4) Mismatch endonuclease mutant Q78R+L123S According to experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the 78th glutamine and the 123rd leucine of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine and serine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 9 and SEQ ID NO: 24, respectively.

[0131] (5) Mismatched endonuclease mutant Q78R+L123T According to experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the 78th glutamine and the 123rd leucine of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine and threonine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 10 and SEQ ID NO: 25, respectively.

[0132] (6) Mismatched endonuclease mutant Q78R+L123M According to experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the glutamine at position 78 and the leucine at position 123 of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine and methionine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 11 and SEQ ID NO: 26, respectively.

[0133] (7) Mismatch endonuclease mutant Q78R+L123C According to experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the 78th glutamine and the 123rd leucine of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine and cysteine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 12 and SEQ ID NO: 27, respectively.

[0134] (8) Mismatched endonuclease mutant Q78R+L125V According to the experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the 78th glutamine and the 125th leucine of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine and valine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 13 and SEQ ID NO: 28, respectively.

[0135] (9) Mismatched endonuclease mutant Q78R+L125A According to the experimental method 1 (1-1), an artificial gene encoding a mutant protein was prepared, wherein the 78th glutamine and the 125th leucine of the polypeptide PF_RS00065 from Pyrococcus furiosus were replaced with arginine and alanine, respectively, to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO: 14 and SEQ ID NO: 29, respectively.

[0136] (10) Heterodimer mismatch endonuclease mutant According to Experimental Method 1 (1-2), artificial genes capable of expressing mutant 1 (S47R+N76D+L123A; SEQ ID NO:7) and mutant 2 (Q78R+L123M; SEQ ID NO:11) as single polypeptides via a linker (SEQ ID NO:35) were prepared to prepare a mismatch endonuclease mutant solution. The amino acid sequence and nucleic acid sequence of the mutant protein are shown in SEQ ID NO:15 and SEQ ID NO:30, respectively.

[0137] Example 2: Evaluation of substrate specificity of mismatch endonuclease mutants Using the mismatch endonuclease mutants prepared in Example 1(1)-(9), and the wild-type mismatch endonuclease (PfuNucS) and its mutant W77F, substrate specificity was evaluated according to Experimental Method 1(2). The results are shown in Table 2.

[0138] [Table 2] Mutant name Substrate specificity GG-specific / TT-specific No mutation (wild type) TT,GG,TG - W77F GG>TG>>TT - S47K+N76D GG GG-specific S47R+N76D+L123A GG GG-specific S47R+N76D+L123G GG GG-specific Q78R+L123S TT TT-specific Q78R+L123T TT TT-specific Q78R+L123M TT TT-specific Q78R+L123C TT TT-specific Q78R+L125V TT TT-specific Q78R+L125A TT TT-specific .

[0139] As shown in Table 2, a GG-specific mismatch endonuclease mutant and a TT-specific mismatch endonuclease mutant having substrate specificity different from that of the wild-type mismatch endonuclease were obtained.

[0140] Example 3: Evaluation of substrate specificity of heterodimeric mismatch endonuclease mutants Using the heterodimeric mismatch endonuclease mutants prepared in Example 1 (10), substrate specificity was evaluated according to Experimental Method 1 (2).

[0141] As a result, the heterodimeric mismatch endonuclease mutant showed GT / TG mismatch-specific substrate specificity, which was different from the substrate specificity of the wild-type mismatch endonuclease and different from the substrate specificity of the mismatch endonuclease mutants prepared in Examples 1(1) to (9).

[0142] Industrial Applicability The present invention can be used in a wide range of fields including genetic technology, biology, medicine and agriculture.

[0143] Sequence Listing Free Text SEQ ID NO: 1: Amino acid sequence of mismatch endonuclease PF0012 from Pyrococcus furiosus SEQ ID NO: 2: Amino acid sequence of mismatch endonuclease PF0012 W77F variant SEQ ID NO: 3: Amino acid sequence of mismatch endonuclease TERMP_01877 from Thermococcus barophilus SEQ ID NO:4: Amino acid sequence of mismatch endonuclease MJ_0225 from Methanococcus jannaschii SEQ ID NO:5: Amino acid sequence of mismatch endonuclease TKO NucS from Thermococcus kodakarensis SEQ ID NO:6: Amino acid sequence of mismatch endonuclease variant S47K+N76D from PF0012 SEQ ID NO:7: Amino acid sequence of the mismatch endonuclease variant S47R+N76D+L123A from PF0012 SEQ ID NO:8: Amino acid sequence of the mismatch endonuclease variant S47R+N76D+L123G from PF0012 SEQ ID NO:9: Amino acid sequence of mismatch endonuclease variant Q78R+L123S from PF0012 SEQ ID NO: 10: Amino acid sequence of mismatch endonuclease variant Q78R+L123T from PF0012 SEQ ID NO: 11: Amino acid sequence of mismatch endonuclease variant Q78R+L123M from PF0012 SEQ ID NO: 12: Amino acid sequence of mismatch endonuclease variant Q78R+L123C from PF0012 SEQ ID NO: 13: Amino acid sequence of mismatch endonuclease variant Q78R+L125V from PF0012 SEQ ID NO: 14: Amino acid sequence of mismatch endonuclease variant Q78R+L125A from PF0012 SEQ ID NO: 15: Amino acid sequence of mismatch endonuclease variant heterodimers from variant S47R+N76D+L123A and variant Q78R+L123M SEQ ID NO: 16: Nucleic acid sequence of the gene encoding the mismatch endonuclease PF0012 from Pyrococcus furiosus SEQ ID NO: 17: Nucleic acid sequence of the gene encoding the mismatch endonuclease PF0012 W77F variant SEQ ID NO: 18: Nucleic acid sequence of the gene encoding the mismatch endonuclease TERMP_01877 from Thermococcus barophilus SEQ ID NO: 19: Nucleic acid sequence of the gene encoding the mismatch endonuclease MJ_0225 from Methanococcus jannaschii SEQ ID NO: 20: Nucleic acid sequence of the gene encoding the mismatch endonuclease TKONucS from Thermococcus kodakarensis SEQ ID NO:21: Nucleic acid sequence of the gene encoding the mismatch endonuclease variant S47K+N76D from PF0012 SEQ ID NO:22: Nucleic acid sequence of the gene encoding the mismatch endonuclease variant S47R+N76D+L123A from PF0012 SEQ ID NO:23: Nucleic acid sequence of the gene encoding the mismatch endonuclease variant S47R+N76D+L123G from PF0012 SEQ ID NO:24: Nucleic acid sequence encoding the gene for the mismatch endonuclease variant Q78R+L123S from PF0012 SEQ ID NO:25: Nucleic acid sequence encoding the gene for the mismatch endonuclease variant Q78R+L123T from PF0012 SEQ ID NO:26: Nucleic acid sequence encoding the gene for the mismatch endonuclease variant Q78R+L123M from PF0012 SEQ ID NO:27: Nucleic acid sequence encoding the gene for the mismatch endonuclease variant Q78R+L123C from PF0012 SEQ ID NO:28: Nucleic acid sequence encoding the gene for the mismatch endonuclease variant Q78R+L125V from PF0012 SEQ ID NO:29: Nucleic acid sequence encoding the gene for the mismatch endonuclease variant Q78R+L125A from PF0012 SEQ ID NO:30: Nucleic acid sequence of the gene encoding the mismatch endonuclease variant heterodimer from variant S47R+N76D+L123A and variant Q78R+L123M SEQ ID NO:31: Synthetic oligonucleotide "NucS-Template-T" SEQ ID NO:32: Synthetic oligonucleotide "NucS-Template-G" SEQ ID NO:33: Synthetic oligonucleotide "NucS-probe-T". The 5' end is labeled with FAM and the 3' end is labeled with Eclipse. SEQ ID NO:34: Synthetic oligonucleotide "NucS-probe-G". The 5' end is labeled with FAM and the 3' end is labeled with Eclipse. SEQ ID NO:35: Amino acid sequence of linker SEQ ID NO: 36: Nucleic acid sequence encoding a linker.

Claims

1. A polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 6 to 8.

2. A nucleic acid encoding the polypeptide according to claim 1.

3. The nucleic acid according to claim 2, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21 to 23.

4. An expression vector comprising the nucleic acid according to claim 2 or 3 and an expression control sequence.

5. A cell transformed with the expression vector according to claim 4, expressing a polypeptide having mismatch endonuclease activity. 6 . A method for cleaving a double-stranded nucleic acid having a mismatch, the method comprising the step of treating the double-stranded nucleic acid with the polypeptide according to claim 1 .

7. A composition comprising the following (a) to (d): (a) the polypeptide according to claim 1; (b) an oligonucleotide which, when hybridized with a non-target nucleic acid, forms a double-stranded nucleic acid comprising at least one mismatch, wherein the double-stranded nucleic acid is cleaved by the polypeptide of (a), and which, when hybridized with a target nucleic acid, forms a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) A polypeptide having DNA polymerase activity.

8. A method for amplifying a nucleic acid, the method comprising the following steps (1) and (2): (1) A step of preparing a composition comprising a nucleic acid molecule as a template and the following (a) to (d): (a) the polypeptide according to claim 1; (b) an oligonucleotide which, when hybridized with a non-target nucleic acid, forms a double-stranded nucleic acid comprising at least one mismatch, wherein the double-stranded nucleic acid is cleaved by the polypeptide of (a), and which, when hybridized with a target nucleic acid, forms a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having DNA polymerase activity; and (2) A step of reacting the composition obtained in step (1) under appropriate conditions to perform nucleic acid amplification. 9 . The method according to claim 8 , wherein the nucleic acid amplification is performed by a polymerase chain reaction (PCR) method, an isothermal nucleic acid amplification method, or a multiple displacement amplification (MDA) method.

10. A method for inhibiting amplification of a nucleic acid comprising a specific nucleotide sequence, the method comprising the step of performing a nucleic acid amplification reaction in the presence of the following (a) to (d): (a) an oligodeoxynucleotide designed to produce one or several mismatches when hybridizing with a nucleic acid comprising a specific nucleotide sequence or its complementary strand; (b) DNA polymerase; (c) at least one pair of oligonucleotide primers; and (d) The polypeptide according to claim 1. The method according to claim 10 , wherein the nucleic acid amplification reaction is polymerase chain reaction (PCR) or isothermal nucleic acid amplification.

12. A non-diagnostic method for preferentially amplifying a target DNA, the method comprising inhibiting the amplification of a DNA having a nucleotide sequence that differs from the target DNA by one or several nucleotides by the method according to claim 10 or 11. 13 . The method according to claim 12 , which is used to amplify one of a DNA having a wild-type nucleotide sequence and a DNA different from the wild-type DNA by a single nucleotide polymorphism mutation different from each other. 14 . The method according to claim 13 , wherein the single nucleotide polymorphism mutation is a single nucleotide polymorphism mutation associated with carcinogenesis or a therapeutic effect of a cancer therapeutic agent.

15. A polypeptide dimer comprising the polypeptide according to claim 1, wherein the dimer is: (1) a homodimer having the polypeptide according to claim 1 as a subunit, or (2) A heterodimer having the polypeptide according to claim 1 and a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 14 as subunits.

Images