Method for detecting a variation in a target base sequence of a nucleic acid, method for selectively inhibiting amplification of a nucleic acid, and kit for carrying out the same

Abstract

The present invention aims to provide a simple method and a kit for implementing the method for detecting mutant genes at a mutant gene content rate lower than the detection limit of mutant genes by the clamp-PCR method. In addition, it aims to provide a simple method and a kit for implementing the method for selectively inhibiting the amplification of a base sequence as a target in a nucleic acid amplification reaction. In a nucleic acid amplification reaction, a diallylamine-sulfur dioxide copolymer containing the following constituent units is used as an enhancer for the inhibition of nucleic acid amplification by clamp nucleic acid: a constituent unit (A) having a structure represented by the general formula (I-a) or the general formula (I-b) or a structure as an acid addition salt thereof: [Chemical 1] #imgabs0# In the formula, R 1 A structural unit (B) having a structure represented by the general formula (I-c) and representing an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group: [Chemical 2] #imgabs1#

Description

[Technical field]

[0001] The present invention relates to a method for detecting a variation in a target base sequence of a nucleic acid in a sample relative to a standard base sequence, a method for selectively inhibiting amplification of a nucleic acid having the target base sequence in a nucleic acid amplification reaction, and a kit for carrying out the same. More specifically, the present invention relates to a method for detecting a variation in a target base sequence relative to a standard base sequence by carrying out nucleic acid amplification in the presence of a specific diallylamine-sulfur dioxide copolymer in a nucleic acid amplification reaction using a clamp nucleic acid (sometimes also referred to as a blocker nucleic acid), a method for selectively inhibiting amplification of a nucleic acid having the target base sequence, and a kit for carrying out the same. [Background technology]

[0002] When a gene mutation has the potential to be a cause of cancer, it is expected that early discovery of such a gene mutation will be associated with early treatment of cancer. In addition, it is known that a mutation in a particular gene is greatly related to the susceptibility to a certain disease, the therapeutic effect of a drug, the strength of side effects, etc. For example, it is known that gefitinib (trade name: Iressa), a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), shows great efficacy in a portion of lung cancer patients with a specific gene mutation, but causes severe interstitial pneumonia in about 0.5% of patients (non-patent document 1). In addition, it is known that in colorectal cancer, if the KRAS gene is mutated, the efficacy of the molecular target drug cetuximab (trade name: Erbitux) is low, and if there is no mutation, the efficacy is high. Therefore, as long as the efficacy or side effects can be predicted for each patient, it becomes possible to control the administration to patients who cannot expect efficacy or patients who are expected to have severe side effects and to conduct effective treatment with fewer side effects.

[0003] It is recognized that understanding gene mutations is important for early detection of cancer and improvement of treatment efficiency, and various technologies for detecting gene mutations are being developed to meet such needs. In particular, significant progress has been seen in gene amplification technologies such as PCR and overall gene analysis technologies such as NGS.

[0004] Of course, the genes extracted from tumor specimens contain a large amount of wild-type genes and a small amount of mutant genes. In the conventional methods for detecting mutant genes, it is still often not sensitive enough to detect a small amount of mutant genes. For example, one of the methods for detecting gene mutations is to non-selectively amplify mutant genes and wild-type genes and then distinguish the mutant genes from the wild-type genes by electrophoresis or hybridization. In this method, it is difficult to detect a very small amount of mutant genes contained in a wild-type gene with sufficient sensitivity and accuracy.

[0005] In contrast, a so-called "clamp method" has been developed, which uses an artificial oligonucleotide having a base sequence complementary to a reference sequence of a wild-type gene to suppress amplification of a wild-type gene and selectively amplify a mutant gene in the gene amplification stage. The artificial nucleic acid used in this method is called a clamp nucleic acid, which has the following characteristics: (i) it strongly hybridizes with a wild-type gene, (ii) it does not strongly hybridize with a mutant gene, and (iii) it is difficult to decompose during nucleic acid amplification. When using it to perform real-time PCR, the frequency of detecting a mutant gene can be changed to 1%, and when using the amplification product of real-time PCR as a template to perform direct sequencing, it is believed that the frequency of detecting a mutant gene can be changed to 0.1% (for example, Patent Document 1, Non-Patent Document 2, Non-Patent Document 3). As sandwich nucleic acids, peptide nucleic acids (PNA), locked nucleic acids (LNA) and bridged nucleic acids (BNA) have been developed, and amplification methods such as the PNA-LNA sandwich PCR method and the BNA sandwich PCR method have been developed (see, for example, Non-Patent Documents 2, 3, 6 and Patent Documents 1 to 9).

[0006] However, even in these clamp methods, when the ratio of mutant genes to wild-type genes in the sample is extremely low, real-time PCR is performed, and the wild-type gene is amplified, but the wild-type gene and the mutant gene may not be distinguished. In addition, direct sequencing using the amplification product of clamp PCR as a template can increase the detection sensitivity of the mutant gene, but sequence determination is not suitable for clinical situations that require on-site processing of a large number of subjects.

[0007] [Prior art literature]

[0008] [Patent Literature]

[0009] [Patent Document 1] Patent No. 6242336

[0010] [Patent Document 2] Patent No. 5813263

[0011] [Patent Document 3] Patent No. 4731324

[0012] [Patent Document 4] Patent No. 4383178

[0013] [Patent Document 5] Patent No. 5030998

[0014] [Patent Document 6] Patent No. 4151751

[0015] [Patent Document 7] Japanese Patent Application Publication No. 2001-89496

[0016] [Patent Document 8] International Publication No. 2003 / 068795

[0017] [Patent Document 9] International Publication No. 2005 / 021570

[0018] [Patent Document 10] Patent No. 3756313

[0019] [Patent Document 11] International Publication No. 2016 / 167320

[0020]

Non-patent literature

[0021] [Non-patent document 1] Thomas J., et al., 2004, The New England Journal of Medicine, 350; 21

[0022] [Non-patent document 2] Nagai K., et al., 2005, Cancer Research, 65: 7276-7282

[0023] [Non-patent document 3] Nishino K. et al., 2019, Manual for detecting EGFR gene mutations in lung cancer patients, version 4.1

[0024] [Non-patent document 4] Luming Z. et al., 2011 BioTechniques, 50: 311-318

[0025] [Non-patent document 5] Nagakubo Y. et al., 2019, BMC Medical Genomics, 12: 162

[0026] [Non-patent document 6] Murina FF et al., 2020, Molecules, 25(4): 786

[0027] [Summary of the Invention]

[0028] [Problems to be solved by the invention]

[0029] Thus, in clinical or research sites, when the ratio of mutant genes to the ratio of wild-type genes in a sample is very low, there is a need for a method for detecting mutant genes, and research on methods corresponding thereto is being promoted (Patent Document 2, Non-Patent Document 4, and Non-Patent Document 5). However, conventional methods require expensive equipment or complicated analysis steps of more than two stages in order to detect mutant genes with high sensitivity (Non-Patent Document 2), and methods that can detect mutant genes in samples with high sensitivity using simple methods are required in clinical sites or research sites.

[0030] Therefore, the present invention aims to provide a simple method and a kit for implementing the method that can detect mutant genes at a mutant gene content rate lower than the detection limit of mutant genes by the conventional clamp method.

[0031] The present invention also aims to provide a simple method for selectively inhibiting amplification of a target base sequence in a nucleic acid amplification reaction and a kit for implementing the method.

[0032]

Methods for solving the problem

[0033] The present inventors have repeatedly studied methods for improving the detection sensitivity of mutant genes by the clamp method. When a nucleic acid amplification reaction is carried out together with a clamp nucleic acid in the presence of a specific diallylamine·sulfur dioxide copolymer, the effect of the clamp nucleic acid that selectively inhibits the amplification of the wild-type gene is enhanced by the presence of the diallylamine·sulfur dioxide copolymer, thereby successfully improving the detection sensitivity of the mutant gene, thereby completing the present invention.

[0034] That is, the present invention provides the following methods, kits, enhancers, and uses.

[0035] [1] A method for detecting a variation in a target base sequence of a nucleic acid in a sample relative to a standard base sequence, comprising performing a nucleic acid amplification reaction using the nucleic acid in the sample as a template using the following substances:

[0036] A nucleic acid comprising a base sequence complementary to the above-mentioned standard base sequence, comprising at least one non-natural nucleotide residue, and

[0037] Diallylamine-sulfur dioxide copolymers containing the following constituent units:

[0038] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0039]

Chemistry 1

[0040]

[0041] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group, and

[0042] The structural unit (B) having a structure represented by the general formula (Ic):

[0043]

Chemistry 2

[0044]

[0045] The presence of the above-mentioned variation is determined based on the total amount of nucleic acid amplification products obtained by the above-mentioned nucleic acid amplification reaction, the number of amplification cycles of the above-mentioned nucleic acid amplification reaction until the total amount of the above-mentioned nucleic acid amplification products reaches a threshold value, the amount of nucleic acid having the above-mentioned variation in the above-mentioned nucleic acid amplification products, or the number of amplification cycles of the above-mentioned nucleic acid amplification reaction until the amount of nucleic acid having the above-mentioned variation in the above-mentioned nucleic acid amplification products reaches a threshold value.

[0046] [2] The detection method described in [1], wherein the above-mentioned diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

[0047] [3] The detection method described in [1], wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

[0048] [4] The detection method according to any one of [1] to [3], wherein the non-natural nucleotide is BNA.

[0049] [5] The detection method according to any one of [1] to [4], wherein the nucleic acid amplification is performed by real-time PCR.

[0050] [6] The detection method according to any one of [1] to [5], wherein the nucleic acid in the sample is genomic DNA.

[0051] [7] The detection method according to any one of [1] to [6], wherein

[0052] When performing the above-mentioned nucleic acid amplification reaction, a nucleic acid amplification reaction using the nucleic acid having the above-mentioned standard base sequence as a template is also performed.

[0053] When the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification product reaches a threshold value when the nucleic acid in the sample is used as a template is smaller than the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification product reaches a threshold value when the nucleic acid having the standard base sequence is used as a template, it is determined that the above-mentioned variation exists in the target base sequence of the nucleic acid in the sample.

[0054] [8] A kit for detecting a variation in a target base sequence of a nucleic acid in a sample relative to a standard base sequence, comprising:

[0055] A nucleic acid having a base sequence complementary to the above-mentioned standard base sequence, containing at least one non-natural nucleotide residue, and

[0056] Diallylamine-sulfur dioxide copolymers containing the following constituent units:

[0057] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0058]

Chemistry 3

[0059]

[0060] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group), and

[0061] The structural unit (B) having a structure represented by the general formula (Ic):

[0062]

Chemistry 4

[0063]

[0064] [9] The kit described in [8], wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

[0065]

[10] The kit described in [9], wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

[0066]

[11] The kit according to any one of [8] to

[10] , wherein the non-natural nucleotide is BNA.

[0067]

[12] A method for inhibiting amplification of a nucleic acid in a sample having a specified target base sequence in a nucleic acid amplification reaction, comprising performing a nucleic acid amplification reaction using the nucleic acid as a template using the following substances:

[0068] A nucleic acid having a base sequence complementary to the target base sequence, containing at least one non-natural nucleotide residue, and

[0069] Diallylamine-sulfur dioxide copolymers containing the following constituent units:

[0070] The structural unit (A) having a structure represented by the general formula (Ia) or (Ib) or a structure of an acid addition salt thereof:

[0071]

Chemistry 5

[0072]

[0073] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group), and

[0074] The structural unit (B) having a structure represented by the general formula (Ic):

[0075]

Chemistry 6

[0076]

[0077]

[13] The method described in

[12] , wherein the above-mentioned diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

[0078]

[14] The method described in

[12] , wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

[0079]

[15] The method according to any one of

[12] to

[14] , wherein the non-natural nucleotide is BNA.

[0080]

[16] A kit for inhibiting amplification of a nucleic acid having a target base sequence in a nucleic acid amplification reaction, comprising:

[0081] A nucleic acid having a base sequence complementary to the target base sequence, containing at least one non-natural nucleotide residue, and

[0082] Diallylamine-sulfur dioxide copolymers containing the following constituent units:

[0083] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0084]

Chemistry 7

[0085]

[0086] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group), and

[0087] The structural unit (B) having a structure represented by the general formula (Ic):

[0088]

Chemistry 8

[0089]

[0090]

[17] The kit described in

[16] , wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

[0091]

[18] The kit described in

[16] , wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

[0092]

[19] The kit according to any one of

[16] to

[18] , wherein the non-natural nucleotide is BNA.

[0093]

[20] A nucleic acid amplification inhibition enhancer that enhances the effect of a sandwich nucleic acid on inhibiting nucleic acid amplification of a nucleic acid having a target base sequence, more specifically, a nucleic acid amplification inhibition enhancer that enhances the effect of a sandwich nucleic acid on inhibiting nucleic acid amplification of a nucleic acid having a target base sequence, wherein the sandwich nucleic acid has a base sequence complementary to the target base sequence and contains at least one non-natural nucleotide residue, and the enhancer contains a diallylamine-sulfur dioxide copolymer, wherein the diallylamine-sulfur dioxide copolymer contains:

[0094] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0095]

Chemistry 9

[0096]

[0097] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group, and

[0098] The structural unit (B) having a structure represented by the general formula (Ic):

[0099]

Chemistry 10

[0100]

[0101]

[21] The enhancer described in

[20] , wherein the above-mentioned diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

[0102]

[22] The enhancer described in

[20] , wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

[0103]

[23] Use of a diallylamine-sulfur dioxide copolymer in the manufacture of a nucleic acid amplification inhibition enhancer for enhancing the nucleic acid amplification inhibition effect of a nucleic acid, wherein the diallylamine-sulfur dioxide copolymer comprises:

[0104] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0105]

Chemistry 11

[0106]

[0107] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group, and

[0108] The structural unit (B) having a structure represented by the general formula (Ic):

[0109]

Chemistry 12

[0110]

[0111]

[24]

[23] The use described in, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

[0112]

[25] The use described in

[23] , wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

[0113]

[26] Use of a diallylamine-sulfur dioxide copolymer for enhancing the inhibitory effect of nucleic acid amplification by sandwich nucleic acid, wherein the diallylamine-sulfur dioxide copolymer comprises:

[0114] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0115]

Chemistry 13

[0116]

[0117] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group, and

[0118] The structural unit (B) having a structure represented by the general formula (Ic):

[0119]

Chemistry 14

[0120]

[0121]

[27]

[26] The use described in, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

[0122]

[28] The use described in

[26] , wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

[0123] The above-mentioned diallylamine-sulfur dioxide copolymer does not inhibit the nucleic acid amplification reaction itself, but can selectively enhance the effect of inhibiting the amplification of nucleic acids having sequences complementary to the sandwich nucleic acids. Therefore, according to one embodiment of the present invention, even in the case where the ratio of nucleic acids having a variation relative to the standard base sequence is very low (for example, less than 0.1%) for nucleic acids having a standard base sequence complementary to the sandwich nucleic acid in the sample, the amplification of nucleic acids having the standard base sequence is selectively inhibited by the sandwich nucleic acid, while nucleic acids having a variation relative to the standard base sequence are amplified normally, and nucleic acids having a variation relative to the standard base sequence can be detected with very high sensitivity by a simple method.

[0124] [Brief description of the attached drawings]

[0125]

Figure 1

[0126]

Figure 2

[0127]

Figure 3

[0128] The embodiments of the present invention will be described in detail. However, the present invention should not be construed as being limited to the following embodiments.

[0129] In one embodiment, the present invention relates to a method for detecting a variation in a target base sequence relative to a standard base sequence by performing nucleic acid amplification in the presence of a specific diallylamine·sulfur dioxide copolymer in a nucleic acid amplification method using a clamp nucleic acid. In other embodiments, the present invention relates to a method for inhibiting the amplification of a nucleic acid having a specified target base sequence by performing a nucleic acid amplification reaction in the presence of a clamp nucleic acid and a specific diallylamine·sulfur dioxide copolymer in a nucleic acid amplification method. In addition, in still other embodiments, the present invention relates to a kit for implementing these methods. In addition, in still other embodiments, the present invention relates to an enhancer of the nucleic acid amplification inhibitory effect of a clamp nucleic acid. Each embodiment is described in detail below.

[0130] [1. Method for detecting a variation in a target base sequence relative to a standard base sequence]

[0131] One embodiment of the present invention relates to a method for detecting a variation in a target base sequence of a nucleic acid in a sample relative to a standard base sequence. This method comprises preparing a nucleic acid having a base sequence complementary to the standard base sequence and containing at least one non-natural nucleotide residue and a specific diallylamine-sulfur dioxide copolymer, and using these to perform a nucleic acid amplification reaction using the nucleic acid in the sample as a template. When the target base sequence has a variation relative to the standard base sequence, the region containing the target base sequence is preferentially amplified over the standard base sequence to detect the presence of the variation.

[0132]

Sample

[0133] The sample provided for the method of the present invention is a sample that is presumed to contain a nucleic acid with a mutation or a nucleic acid to be inhibited from amplification, corresponding to the embodiment. In the present embodiment, it is a sample that is presumed to contain a nucleic acid for which a mutation is desired to be detected. The sample is, for example, a sample prepared from a subject collected from a mammal (typically a human). For example, a sample prepared from a liquid sample such as blood, pleural effusion, bronchial washings, bone marrow fluid, lymph fluid, or a solid sample such as a lymph node, blood vessel, bone marrow, brain, spleen, skin, etc. can be cited. Since the method of the present invention can detect a mutation in a nucleic acid with very high sensitivity, not only a sample from a subject collected from a lesion such as a cancer, but also a sample from a subject of a mutant gene containing only a trace amount of blood can be used.

[0134]

Target base sequence

[0135] In the present application, "target base sequence" refers to a base sequence consisting of one or more bases that is the object of variation detection or amplification inhibition according to the method according to the embodiment of the present invention. In addition, "nucleic acid in a sample" is a nucleic acid that may contain a base sequence that is the object of variation detection or amplification inhibition. However, "target base sequence" does not refer to a base sequence that actually has a variation or actually contains a base sequence that is inhibited by amplification, and "nucleic acid in a sample" does not refer to a base sequence that actually contains a base sequence that is the object of variation detection or amplification inhibition.

[0136] In the method according to this embodiment, the "target base sequence" is a base sequence consisting of one or more bases that is the object of detecting the presence or absence of a mutation, and the "nucleic acid in the sample" is a nucleic acid containing the base sequence that is the object of detecting the presence or absence of a mutation. In addition, the "region containing the target base sequence" is a region that becomes the target of amplification by the method of the present invention.

[0137]

Nucleic acid in the sample

[0138] Nucleic acid may be DNA or RNA, and may include all natural or non-natural nucleic acids such as genomic DNA, cDNA, plasmid DNA, cell-free DNA, circulating DNA, RNA, miRNA, mRNA, etc. When using a sample from a subject collected from a mammal (typically a human), the nucleic acid that becomes the object of mutation detection is often DNA, and typically, it is genomic DNA of great clinical significance.

[0139] As genomic DNA, for example, the genomic DNA associated with the onset and / or treatment sensitivity of a specific disease can be enumerated. Such diseases are mostly known, and their representative examples are various cancers. It is also known that as the gene associated with the onset and / or treatment sensitivity of cancer, for example, ABL / BCR fusion gene (chronic myeloid leukemia), HER2 gene (breast cancer), EGFR gene (non-small cell lung cancer), c-KIT gene (digestive tract stromal tumor), KRAS gene (colorectal cancer, pancreatic cancer), BRAF gene (melanoma, colorectal cancer), PI3KCA gene (lung cancer, colorectal cancer), FLT3 gene (acute myeloid leukemia), MYC gene (various cancers), MYCN gene (neuroblastoma), MET gene (lung cancer, gastric cancer, melanoma), BCL2 gene (cell B lymphoma) and EML4 / ALK fusion gene (lung cancer) can be enumerated.

[0140]

Standard base sequence and variation

[0141] In the present application, a "standard base sequence" refers to a base sequence that serves as a reference for determining whether the "target base sequence" has a variation in a method for detecting a variation in the "target base sequence", and a "variation" refers to a state in which the "target base sequence" has a certain base sequence difference relative to the "standard base sequence" due to substitution, insertion, deletion, inversion, duplication, transposition, or a combination of two or more of these. In the present application, such a "variation" may be a base sequence difference of less than 20%, a base sequence difference of less than 10%, a base sequence difference of less than 5%, or a base sequence difference of less than 1%.

[0142] The "standard base sequence" can be selected from various base sequences corresponding to the purpose of the inspection. Typically, it is a base sequence derived from a wild-type nucleic acid, preferably a base sequence derived from a wild-type gene known to be associated with the onset and / or treatment sensitivity of a specific disease. Thus, the "variant nucleic acid" is typically a nucleic acid that has a certain base sequence difference relative to the base sequence of the nucleic acid derived from the wild type by substitution, insertion, deletion, inversion, duplication, transposition or a combination of two or more of these, preferably a nucleic acid that has such a base sequence difference relative to the wild-type gene known to be associated with the onset and / or treatment sensitivity of a specific disease. For example, it is known that the variation of the 12th codon or the 13th codon of the 2nd exon of the KRAS gene is associated with the efficacy of the molecular target drug cetuximab. In addition, it is known that in the BRAF gene, the variation of the 600th codon of the 15th exon is associated with the efficacy of the molecular target drug cetuximab or panitumumab.

[0143] Since the specific diallylamine-sulfur dioxide copolymer particularly increases the inhibitory effect of nucleic acid amplification by the sandwiched nucleic acid, the expected value of the ratio of the target base sequence containing the above mutation relative to the total of the target sequence (standard base sequence) not containing the mutation and the target sequence containing the above mutation contained in the nucleic acid in the above sample can also be less than 0.1%. In addition, in a preferred embodiment, it can be less than 0.05%, and in a more preferred embodiment, it can also be less than 0.01%. Among them, the expected value of the ratio of the target base sequence containing the mutation is clinically required for each mutation.

[0144]

Nucleic acid clamp

[0145] In the present application, "sandwich nucleic acid" refers to an artificial nucleic acid composed of an oligonucleotide containing at least one non-natural nucleotide having a base sequence complementary to a "base sequence" (or "standard base sequence" or "target base sequence" depending on the embodiment) that is a target for inhibiting nucleic acid amplification. "Sandwich nucleic acid" hybridizes with the "base sequence" that is a target for inhibiting nucleic acid amplification to inhibit nucleic acid amplification, but does not strongly hybridize with a nucleic acid having a base sequence that is not complementary to the base sequence to inhibit nucleic acid amplification.

[0146] In the method for detecting a variation in a "target base sequence", the "base sequence" that is the target of nucleic acid amplification inhibition by the "sandwich nucleic acid" is the "standard base sequence", and the "sandwich nucleic acid" hybridizes with the "standard base sequence" to inhibit its nucleic acid amplification.

[0147] Examples of non-natural nucleotides include peptide nucleic acids (PNA), bridged nucleic acids (BNA), peptide nucleic acids with phosphate groups (PHONA), and morpholino nucleic acids. In addition, first-generation BNAs are also called locked nucleic acids (LNA) (see, for example, Non-Patent Documents 2 and 3 and Patent Documents 1 and 2).

[0148] A PNA-containing clamp nucleic acid (PNA clamp) is a fully modified artificial nucleic acid composed of an oligonucleotide containing one or more nucleotides whose backbone is formed by a peptide bond instead of a phosphate bond of a nucleic acid. The modified nucleotide typically replaces the sugar-phosphate backbone of the nucleotide with a backbone having N-(2-aminoethyl)glycine as a unit, and has a structure in which a base is bound by a methylene carbonyl group. Details of PNA are described in non-patent literature 6. Since the hybridization ability of the PNA clamp with a complementary DNA chain is stronger than that of natural DNA, it is not decomposed by nucleases and has preferred characteristics as a clamp nucleic acid.

[0149] BNA-containing clamp nucleic acid (BNA clamp) is an artificial nucleic acid composed of an oligonucleotide containing one or more non-natural nucleotides having a structure that cross-links the oxygen atom at the 2' position and the carbon atom at the 4' position of the ribose. The first generation of BNA is also called locked nucleic acid (LNA). The BNA clamp has a fixed helical structure and can form a very stable double-stranded chain by hybridizing with a nucleic acid with a complementary chain. In addition, since the binding to the nucleic acid with a complementary chain is strengthened in proportion to the number of modified nucleotides, the appropriate hybridization energy can be obtained by adjusting the length of the nucleic acid and the number of modified nucleotides, and the clamp effect can be exerted with a relatively short oligonucleotide chain.

[0150] A representative example of a BNA clamp is an oligonucleotide comprising a modified nucleotide having the following structure.

[0151]

Chemistry 15

[0152]

[0153] First generation BNA: 2',4'-BNA (LNA) (e.g., Patent Document 10)

[0154]

Chemistry 16

[0155]

[0156]

Chemistry 17

[0157]

[0158]

Chemistry 18

[0159]

[0160] (Where,

[0161] Base represents a pyrimidine or purine nucleic acid base or a hydroxyl group, a hydroxyl group protected by a protecting group synthesized by nucleic acid, an alkoxy group having 1 to 5 carbon atoms, a thiol group, a thiol group protected by a protecting group synthesized by nucleic acid, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected by a protecting group synthesized by nucleic acid, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, a pyrimidine or purine nucleic acid base substituted by a substituent selected from the group consisting of an alkyl group having 1 to 5 carbon atoms and a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom or an iodine atom),

[0162] R2 represents a hydrogen atom, a linear or branched alkyl group having 1 to 20 carbon atoms, a linear or branched alkenyl group having 2 to 20 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms, an aryl group having 6 to 14 carbon atoms, an aralkyl group in which an alkyl group having 1 to 6 carbon atoms is substituted with an aryl group having 6 to 14 carbon atoms, an acyl group, a sulfonyl group, a silyl group, or a labeling molecule,

[0163] m is an integer from 0 to 2,

[0164] n is an integer from 1 to 3)

[0165] The modified nucleotide of formula (IV) is preferably Base, selected from benzoylaminopurine-9-yl, adenine, 2-isobutyrylamino-6-hydroxypurine-9-yl, guanine, 2-oxo-4-benzoylamino-1,2-dihydropyrimidine-1-yl, cytosine, 2-oxo-5-methyl-4-benzoylamino-1,2-dihydropyrimidine-1-yl, 5-methylcytosine, uracil, and thymine, R1 is methyl, m is 0, and n is 1.

[0166] However, the BNA used in the present invention is not limited to the above, and other BNAs (e.g., 5'-amino-2',4'-BNA, 2',4'-BNACOC, 2',4'-BNANC, etc.) can also be used. In addition, in the BNA clamp, the bond between the modified nucleotide and the nucleotide, or between the modified nucleotides is usually a phosphodiester bond, and may contain a phosphorothioate bond. The details of BNA and the BNA clamp are described in Patent Documents 1 to 10, and those skilled in the art of the technical field to which the present invention belongs can easily understand which artificial nucleic acid corresponds to the BNA clamp from the known technical matters in these documents, etc.

[0167] As a clamp nucleic acid, a BNA clamp is preferred, and a BNA clamp containing a modified nucleotide having a structure of the special formula (IV) is preferred, in that a relatively small number of nucleotides can form a stable double-stranded chain with a nucleic acid of a complementary sequence and selectively inhibit the nucleic acid amplification of the nucleic acid (when a variation detection method using the amplification cycle number of a wild-type gene as a benchmark is used, such as the intercalator method described later, the ΔΔΔCt value is large).

[0168] The length of the sandwich nucleic acid and the number of non-natural nucleotides are factors that determine the binding force to the nucleic acid that is the object of inhibiting nucleic acid amplification. Therefore, these factors are preferably set appropriately in a manner that can strongly bind to the nucleic acid that is the object of inhibiting nucleic acid amplification (a nucleic acid with a standard base sequence in the method of detecting mutations) and selectively inhibit the amplification of the nucleic acid. In this regard, the length of the sandwich nucleic acid is usually 5 to 50mer, preferably 6 to 30mer, more preferably 7 to 25mer, more preferably 8 to 20mer, and particularly preferably 9 to 15mer.

[0169] From the same point of view, the ratio of the non-natural nucleic acid in the sandwich nucleic acid is usually 10 to 100%, preferably 30 to 95%, more preferably 40 to 90%, further preferably 50 to 85%, and particularly preferably 60 to 80%.

[0170] The synthesis of the sandwich nucleic acid can be performed based on the documents of Patent Documents 7 to 10. Alternatively, the synthesis of the sandwich nucleic acid can be outsourced to the deposited company.

[0171]

Diallylamine·Sulfur dioxide copolymer

[0172] In the method of the present invention, when a nucleic acid amplification reaction is performed using a nucleic acid in a sample as a template, the nucleic acid amplification reaction is performed in the presence of a diallylamine-sulfur dioxide copolymer having a specific structure together with the above-mentioned sandwich nucleic acid. The diallylamine-sulfur dioxide copolymer used in the present invention is a copolymer that does not inhibit nucleic acid amplification reaction itself but enhances the selective nucleic acid amplification inhibition effect of the sandwich nucleic acid, and the copolymer contains:

[0173] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0174]

Chemistry 19

[0175]

[0176] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group, and

[0177] The structural unit (B) having a structure represented by the general formula (Ic):

[0178]

Chemistry 20

[0179]

[0180] The diallylamine-sulfur dioxide copolymer is a copolymer containing the above-mentioned structural unit (A) and the above-mentioned structural unit (B), and thus may be composed of only the structural unit (A) and the structural unit (B), or may optionally have other structural units in addition to the structural unit (A) and the structural unit (B). Examples of other structural units include various cationic structural units other than the structural unit (A), various nonionic structural units other than the structural unit (B), and various anionic structural units.

[0181] The total ratio of the structural units (A) and the structural units (B) to the total structural units of the diallylamine-sulfur dioxide copolymer is not particularly limited, but is usually 80 mol% or more, preferably 80 to 100 mol%, more preferably 90 to 100 mol%, and particularly preferably 95 to 100 mol%.

[0182]

Constitutional unit (A)

[0183] The structural unit (A) constituting the diallylamine-sulfur dioxide copolymer used in the present invention is a diallylamine-based structural unit having the above-mentioned specific structure.

[0184] The diallylamine-sulfur dioxide copolymer may contain only one constituent unit (A) or two or more constituent units (A). When containing two or more constituent units (A), the ratio of the sum of the constituent units (A) and the constituent unit (B) in the total constituent units of the diallylamine-sulfur dioxide copolymer or the ratio of the constituent units (A) and the constituent unit (B) is calculated based on the total amount of the two or more constituent units (A).

[0185] The structural unit (A) has a structure represented by the general formula (Ia) or the general formula (Ib) or a structure as an acid addition salt thereof:

[0186]

Chemistry 21

[0187]

[0188] In the general formula (Ia) and the general formula (Ib), R 1 It represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group.

[0189] Since no matter what option is chosen, R 1 It becomes a group having at least one carbon atom, and structural unit (A) becomes a structural unit having a structure containing a tertiary amino group.

[0190] R 1 An alkyl group having 5 or less carbon atoms or a benzyl group is preferred, a methyl group or an ethyl group is more preferred, and a methyl group is particularly preferred.

[0191] The structural unit (A) may have a structure such as an inorganic acid salt or an organic acid salt of the structure represented by the above structural formula (1-a) or (1-b), that is, a structure as an acid addition salt.

[0192] When producing a diallylamine-sulfur dioxide copolymer, it is preferred to use a diallylamine monomer having an addition salt to introduce the constituent unit (A) from the viewpoint of production cost, etc. Since the process of removing the addition salt of HCl or the like from the polymer is cumbersome and also causes an increase in cost, it is also a preferred embodiment from the viewpoint of cost, etc. to use the constituent unit (A) in the form of an addition salt that can be produced without requiring such a process.

[0193] From the viewpoint of availability and controllability of the reaction, the inorganic acid salt or organic acid salt in the structural unit (A) of this embodiment is preferably a hydrochloride, a carboxylate, a sulfonate, or an alkyl sulfate ester salt, and particularly preferably a hydrochloride.

[0194] For example, the name of diallylamine·sulfur dioxide copolymer is also presented, and the structural unit (A) is usually derived from a diallylamine monomer.

[0195] More specific examples of suitable structural units (A) include structural units derived from diallylmethylamine and hydrochlorides thereof, structural units derived from diallylethylamine and hydrochlorides thereof, and the like.

[0196] The proportion of the structural unit (A) in the total structural units of the diallylamine-sulfur dioxide copolymer is usually 40 mol% or more, preferably 50 to 80 mol%, particularly preferably 50 to 70 mol%. When two or more structural units (A) are contained, the above proportion is defined based on the total amount of the two or more structural units (A).

[0197]

Constituent unit (B)

[0198] The constituent unit (B) that constitutes the diallylamine-sulfur dioxide copolymer used in the present invention is a constituent unit having a structure represented by the general formula (I-c):

[0199]

Chemical formula 22

[0200]

[0201] Regarding the method, kit, and nucleic acid amplification inhibition by clamping nucleic acids of the present invention, in a polymer composed of only the constituent unit (A), the stronger the cationic charge, the more the PCR reaction is inhibited, and the stronger the binding between the nucleic acids forming a double strand. In addition to the above-mentioned constituent unit (A), in the diallylamine-sulfur dioxide copolymer having the constituent unit (B), the constituent unit (B) moderately interferes with the cationic charge of the constituent unit (A). Therefore, a mutant gene can be detected at a mutant gene content rate lower than the detection limit of the mutant gene by the sandwich method, and the amplification of the target base sequence can be selectively inhibited in the nucleic acid amplification reaction.

[0202] As the name of the diallylamine-sulfur dioxide copolymer also indicates, the constituent unit (B) is usually derived from sulfur dioxide.

[0203] The proportion of the constituent unit (B) in the total constituent units of the diallylamine-sulfur dioxide copolymer is preferably 10 mol% or more.

[0204] The proportion of the constituent unit (B) is more preferably 20 to 50 mol%, and particularly preferably 30 to 50 mol%.

[0205] The proportions of the constituent unit (A) and the constituent unit (B) in the diallylamine-sulfur dioxide copolymer are not particularly limited and can be appropriately set according to the relationship with the object of the present invention. In addition, as long as they can be copolymerized, they can be set to any proportion. From the viewpoint of increasing the molecular weight of the diallylamine-sulfur dioxide copolymer, the proportions of the two constituent units are preferably not very different. For example, the molar ratio of the constituent unit (A) to the constituent unit (B) is preferably 90:10 to 40:60, more preferably 80:20 to 50:50, and particularly preferably 70:30 to 50:50.

[0206] The molar ratio of the constituent unit (A) to the constituent unit (B) in the diallylamine-sulfur dioxide copolymer can be determined by reprecipitating the diallylamine-sulfur dioxide copolymer with an organic solvent such as isopropyl alcohol or acetone, and analyzing the reprecipitate using a Perkin Elmer 2400IICHNS / O fully automatic elemental analyzer or an apparatus having equivalent performance in an appropriate mode corresponding to the structure of the above-mentioned constituent unit, when the structure of the constituent unit (A) is known. Furthermore, the measurement can be carried out by using helium gas as a carrier gas, measuring a solid sample in a tin capsule, dropping it in a combustion tube, and burning the sample in a pure oxygen gas at a combustion temperature of 1800°C or more, detecting each measurement component by a head-on chromatography method using a separation column and a thermal conductivity detector, and quantitatively determining the content of each element using a correction factor. In addition, when the structure of the constituent unit (A) in the diallylamine-sulfur dioxide copolymer is unknown, the structure of the constituent unit (A) can be determined by a known method using 1H-NMR or 13C-NMR before the measurement using the above-mentioned elemental analyzer. Alternatively, it can be calculated from the amount of each monomer supplied during the production (copolymerization) of the diallylamine-sulfur dioxide copolymer and the amount of each monomer remaining without collecting the diallylamine-sulfur dioxide copolymer. In addition, since the ratio (molar ratio) of the constituent units derived from each monomer in the diallylamine-sulfur dioxide copolymer is roughly the same as the feed composition (molar ratio) of each constituent unit, in this specification, the mixing ratio of the monomers is conveniently treated as the ratio (molar ratio) of the constituent units.

[0207] Taking the above into consideration, preferred diallylamine·sulfur dioxide copolymers are diallylmethylamine·sulfur dioxide copolymers, diallylmethylamine acid addition salt·sulfur dioxide copolymers, diallylethylamine·sulfur dioxide copolymers, and diallylethylamine acid addition salt·sulfur dioxide copolymers, and particularly preferred are diallylmethylamine·sulfur dioxide copolymers and diallylmethylamine acid addition salt·sulfur dioxide copolymers.

[0208] The diallylamine-sulfur dioxide copolymer may have another structural unit in addition to the above-mentioned structural unit (A) and structural unit (B).

[0209] Examples of other structural units include various cationic structural units other than the structural unit (A), various nonionic structural units other than the structural unit (B), and various anionic structural units.

[0210] [(C) Cationic structural unit other than structural unit (A)]

[0211] Examples of the various cationic structural units (C) other than the structural unit (A) include diallylamine structural units other than the structural unit (A), for example, diallylamine structural units having a secondary amino group or a quaternary amino group, or addition salts thereof, various monoallylamine-based structural units or addition salts thereof, and cationic methacrylamide-based structural units.

[0212] Among them, a diallylamine constitutional unit having a secondary amino group can be preferably used.

[0213] When the diallylamine-sulfur dioxide copolymer has a cationic structural unit other than the structural unit (A), the content of the cationic structural unit is not particularly limited. The appropriate amount varies depending on the type of the cationic structural unit, but is preferably 20 mol% or less, particularly preferably 0 to 10 mol%.

[0214] [(D) Anionic structural unit]

[0215] The anionic structural unit (D) that can be introduced as a structural unit other than the structural units (A) and (B) is not particularly limited as long as it is a structural unit having an anionic group such as a carboxyl group. Preferred examples include structural units derived from unsaturated dicarboxylic acid monomers such as maleic acid and fumaric acid, and structural units derived from unsaturated monocarbon monomers such as acrylic acid and methacrylic acid. Among them, maleic acid and acrylic acid can also be preferably used.

[0216] When the diallylamine-sulfur dioxide copolymer has anionic structural units, the content of the anionic structural units other than the structural unit (B) is not particularly limited. The appropriate amount varies depending on the type of the anionic structural unit, but is preferably 20 mol% or less, particularly preferably 0 to 10 mol%.

[0217] From the viewpoint of adjusting the cationic load of the constituent unit (A), the anionic constituent unit (D) may be contained within the above-mentioned range when the cationic load of the constituent unit (A) is too large, but is preferably not contained when the cationic load of the constituent unit (A) is moderate.

[0218] [(E) Nonionic structural unit other than structural unit (B)]

[0219] The nonionic constituent unit (E) other than the constituent unit (B) in the present embodiment may be a constituent unit derived from a nonionic monomer other than sulfur dioxide copolymerizable with the constituent unit (A) and the anionic constituent unit (B), and there is no particular limitation other than this. It is preferred to use constituent units derived from methacrylate monomers, acrylate monomers, methacrylamide monomers, acrylamide monomers, etc. As more specific examples, constituent units derived from methyl methacrylate, ethyl methacrylate, butyl methacrylate, methyl acrylate, ethyl acrylate, butyl acrylate, methacrylamide, N-methylmethacrylamide, dimethylmethacrylamide, N-(3-dimethylaminopropyl)methacrylamide, acrylamide, dimethylacrylamide, hydroxyethylacrylamide, dimethylaminopropylacrylamide, dimethylaminopropylacrylamide methyl chloride quaternary salt, acryloylmorpholine, isopropylacrylamide, 4-t-butylcyclohexylacrylate, etc. can be cited. Among them, it is particularly preferred to use constituent units derived from acrylamide, hydroxyethylacrylamide or dimethylacrylamide.

[0220] The nonionic structural unit other than the structural unit (B) can be introduced into the polymer usually by using a nonionic monomer as a monomer.

[0221] When the diallylamine-sulfur dioxide copolymer has a nonionic structural unit other than the structural unit (B), the content of the nonionic structural unit other than the structural unit (B) is not particularly limited. The appropriate amount varies depending on the type of the nonionic structural unit, but is preferably 20 mol% or less, particularly preferably 0 to 10 mol%.

[0222] There is no particular limitation on the molecular weight of the diallylamine·sulfur dioxide copolymer, and a diallylamine·sulfur dioxide copolymer having an appropriate molecular weight may be obtained or polymerized according to the usage form in the method, kit, etc. of the present invention or the relationship with other components.

[0223] From the viewpoint of the ease of forming a reaction solution with nucleic acid or the like or polymerizing at a practically acceptable time and cost, the weight average molecular weight (Mw) of the diallylamine-sulfur dioxide copolymer is preferably 300 to 30000. The weight average molecular weight (Mw) is more preferably 1000 to 9000. The weight average molecular weight (Mw) is more preferably 1500 to 6000, and particularly preferably 2000 to 4000.

[0224] The weight average molecular weight (Mw) of the diallylamine·sulfur dioxide copolymer can be measured, for example, by gel permeation chromatography (GPC method) using a liquid chromatograph.

[0225] Specifically, for example, the weight average molecular weight can be determined by the following method using a Hitachi L-6000 high performance liquid chromatograph. As an eluent flow pump, a Hitachi L-6000 is used, as a detector, a Shodex RI-101 differential refractive index detector is used, and as a column, a Shodex Asahipak water-based gel filtration type GS-220HQ (excluding a limiting molecular weight of 3,000) and a GS-620HQ (excluding a limiting molecular weight of 2 million) are connected in series. The sample is modulated to a concentration of 0.5 g / 100 ml with an eluent, and 20 μl is used. In the eluent, a 0.4 mol / L sodium chloride aqueous solution is used. The column temperature is 30° C. and the flow rate is 1.0 ml / min. A calibration curve was obtained using polyethylene glycols having molecular weights of 106, 194, 440, 600, 1470, 4100, 7100, 10300, 12600, 23000, etc. as standard substances, and the weight average molecular weight (Mw) of the copolymer was obtained based on the calibration curve.

[0226] The molecular weight of the diallylamine-sulfur dioxide copolymer can be appropriately adjusted by adjusting the type and composition of monomers contributing to the polymerization, the temperature, hours and pressure in the polymerization step, the type and amount of the radical initiator used in the polymerization step, and the like.

[0227] The rotational viscosity [η] of the diallylamine·sulfur dioxide copolymer is also not particularly limited and can be appropriately set in consideration of the use form or manufacturing cost in the method, kit, etc. of the present invention. In the state of a 25.0 mass % aqueous solution, it is preferably 1.0 to 150.0 mPa·s (25°C), more preferably 1.5 to 30.0 mPa·s (25°C), and particularly preferably 2.0 to 20.0 mPa·s (25°C).

[0228] The rotational viscosity [η] can be measured by a conventional method in the art, for example, by a digital B-type viscometer DV-3T manufactured by AMETEK Brookfield. When measuring, a ULA aptamer can be used, and typically the liquid volume is 16 mL and the liquid temperature is 25°C.

[0229] The rotational viscosity [η] can also be appropriately adjusted by adjusting the dilution concentration during polymerization, the type and composition of monomers contributing to polymerization, the temperature, hours and pressure in the polymerization step, the type and amount of the radical initiator used in the polymerization step, etc.

[0230] [Method for producing diallylamine-sulfur dioxide copolymer]

[0231] The method for producing the diallylamine-sulfur dioxide copolymer is not particularly limited and can be produced by a method known in the art. For example, it can be produced by copolymerizing a diallylamine monomer having a structure corresponding to the constituent unit (A), a monomer corresponding to the constituent unit (B), which is usually sulfur dioxide, and a monomer having a structure corresponding to other constituent units as desired.

[0232] The solvent used in copolymerizing the diallylamine monomer and sulfur dioxide is not particularly limited and may be an aqueous solvent or an organic solvent such as alcohol, ether, sulfoxide, or amide, but is preferably an aqueous solvent.

[0233] The monomer concentration when copolymerizing diallylamine monomers and sulfur dioxide varies depending on the type of monomer and the type of solvent used for the copolymerization. In the case of an aqueous solvent, it is usually 10 to 75% by mass. This copolymerization reaction is usually a free radical polymerization reaction and is carried out in the presence of a free radical polymerization catalyst. The type of free radical polymerization catalyst is not particularly limited, and preferred examples thereof include peroxides such as t-butyl hydroperoxide, persulfates such as ammonium persulfate, sodium persulfate, potassium persulfate, and water-soluble azo compounds such as azobis-based and diazo-based.

[0234] The amount of the radical polymerization catalyst added is generally 0.1 to 20 mol%, preferably 1.0 to 10 mol%, relative to the total monomers. The polymerization temperature is generally 0 to 100° C., preferably 5 to 80° C., and the polymerization time is generally 1 to 150 hours, preferably 5 to 100 hours. The polymerization atmosphere does not cause a major problem in polymerization in the air, and can also be carried out in an atmosphere of an inert gas such as nitrogen.

[0235]

Nucleic acid amplification reaction

[0236] In the method according to the present embodiment, a nucleic acid amplification reaction is performed using a nucleic acid in a sample as a template, using a sandwich nucleic acid and a specific diallylamine-sulfur dioxide copolymer.

[0237] The nucleic acid amplification method is not particularly limited, and examples thereof include real-time PCR, digital PCR, conventional PCR, PCR methods such as RT-PCR, LAMP, ICAN, NASBA, LCR, SDA, TRC, and TMA. The nucleic acid amplification method is preferably PCR from the perspective of the versatility and simplicity of the method, and more preferably real-time PCR from the perspective of easy quantification of nucleic acid amplification products.

[0238] The composition of the reaction solution for nucleic acid amplification varies somewhat depending on the nucleic acid amplification method used. Generally, in addition to the above-mentioned nucleic acid and diallylamine·sulfur dioxide copolymer, a primer set, deoxynucleoside triphosphates as substrates (for example, dATP, dTTP, dCTP and dGTP: hereinafter collectively referred to as dNTP) and a nucleic acid synthetase (for example, DNA polymerase) are contained in the buffer. Depending on the situation, magnesium ions may also be contained as a cofactor for the nucleic acid synthetase (for example, contained in the reaction solution at a concentration of 1 to 6 mM).

[0239] When a nucleic acid amplification method such as real-time PCR is used that simultaneously performs nucleic acid amplification and detection of the amplified nucleic acid, appropriate detection reagents such as intercalators, fluorescent labeled probes, and circulating probes are also contained in the nucleic acid amplification reaction solution.

[0240] The primer set may be any one that can amplify a region containing the target base sequence by a nucleic acid amplification method, and is usually designed to hybridize with a designated region adjacent to the region to be amplified. The primer may contain a sequence that hybridizes with a portion of the target base sequence.

[0241] Primers can be composed of nucleic acids such as DNA and RNA. The concentration of primers is usually selected in the range of 20nM to 2μM in the reaction solution. The length of the primer is usually 5 to 40mer, preferably 12 to 35mer, more preferably 14 to 30mer, and more preferably 15 to 25mer. The distance between primers, that is, the region to be amplified is usually 50 to 5000 bases, preferably 100 to 2000 bases. The design of primers can be carried out according to the manual, or appropriate software for primer design can be used. As such software, for example, Primer3 software (http: / / frodo.wi.mit.edu) etc. can be cited.

[0242] The concentration of dNTP is usually selected from dATP, dTTP, dCTP and dGTP, and the concentration of each in the reaction solution is 100 to 400 μM. As nucleic acid synthetase, for example, DNA polymerase, RNA polymerase, reverse transcriptase, etc. can be cited, as long as the enzyme with suitable properties is used corresponding to the nucleic acid amplification method used. For example, when it is a PCR method, TaqDNA polymerase, PfuDNA polymerase, and various heat-resistant DNA polymerases developed by various companies associated with Bioscience are preferably used.

[0243] The buffer preferably has the optimum pH and the optimum salt concentration of the DNA polymerase used. The nucleic acid amplification method may also contain ribonuclease H (RNase H) or reverse transcriptase (RT) etc. Furthermore, since various test kits are commercially available for each nucleic acid amplification method, the nucleic acid amplification reaction solution may also be used with a test kit attached to such a commercially available test kit.

[0244] The concentration of the nucleic acid that serves as a template for the nucleic acid amplification reaction, i.e., the "nucleic acid in the sample", in the reaction solution is usually set to 0.3 ng to 3 μg per 100 μl of reaction solution. If the amount of nucleic acid that serves as a template is too large, the frequency of nonspecific amplification increases, so it is preferably suppressed to less than 0.5 μg per 100 μl of reaction solution.

[0245] As the amount of the sandwich nucleic acid in the reaction solution, it is preferred to select an amount corresponding to the amount of the nucleic acid having the target base sequence estimated to have a mutation relative to the amount of the nucleic acid having the standard base sequence (e.g., wild-type nucleic acid) in the sample, so that when the target base sequence has a mutation, the amplification of the nucleic acid having the standard base sequence is sufficiently suppressed and the nucleic acid containing the target base sequence having a mutation is preferentially amplified. Since the wild-type nucleic acid is usually the majority relative to the mutant nucleic acid in the sample, it is preferably 10 nM to 1 μM, more preferably 20 nM to 500 nM, and particularly preferably 50 nM to 200 nM.

[0246] The concentration of the diallylamine·sulfur dioxide copolymer in the reaction solution is preferably selected to a suitable concentration in a manner that has no adverse effect on the nucleic acid amplification reaction and enhances the selective inhibition effect of the nucleic acid amplification reaction by the nucleic acid. The concentration of the diallylamine·sulfur dioxide copolymer in the reaction solution also varies depending on the type of polymer. From this point of view, it is usually set to 0.001 to 0.500 mass %, preferably 0.005 to 0.100 mass %, more preferably 0.010 to 0.050 mass %, and particularly preferably 0.020 to 0.030 mass %.

[0247] When the above-mentioned sandwich nucleic acid and diallylamine·sulfur dioxide copolymer are used to carry out a nucleic acid amplification reaction using the nucleic acid in the sample as a template, the sandwich nucleic acid strongly binds to the nucleic acid having the standard base sequence, and the annealing of the primers and / or the extension of the primers during the amplification reaction are inhibited. The coexisting diallylamine·sulfur dioxide copolymer enhances this inhibitory effect. On the one hand, since the binding force of the sandwich nucleic acid is greatly reduced for the base sequence having a variation relative to the standard base sequence, the annealing and the extension of the primer are hardly inhibited, and the normal nucleic acid amplification reaction occurs. In addition, the diallylamine·sulfur dioxide copolymer itself does not inhibit the nucleic acid amplification reaction. As a result, according to the method of this embodiment, even if the nucleic acid having a variation is a trace amount (for example, less than 0.1% or less than 0.05%, especially less than 0.02%) among the nucleic acids in the sample, in the case where the nucleic acid having the standard base sequence dominates, the amplification of the nucleic acid having the standard base sequence is selectively inhibited, and the nucleic acid having a variation is preferentially amplified, so that the detection of the nucleic acid having a variation contained in an extremely trace amount becomes possible.

[0248] In the method of this embodiment, the presence of a mutation is detected by utilizing the characteristics of the above-mentioned nucleic acid amplification reaction. That is, since the amplification of a nucleic acid having a standard base sequence is selectively suppressed, when the target base sequence has a mutation relative to the standard base sequence, the region containing the target base sequence is preferentially amplified. As a result, the speed of the nucleic acid amplification reaction, the amount of the reaction product, etc., differ depending on the presence or absence of the mutation, and these are utilized in the detection of the mutation.

[0249] In this embodiment, the detection of the variation may include a method of detecting the final product of the amplification reaction and a method of confirming the amplification over time during the amplification reaction, for example, based on the total amount of the obtained nucleic acid amplification product, the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification product reaches a threshold value, the amount of the nucleic acid having the variation in the nucleic acid amplification product, or the number of amplification cycles of the nucleic acid amplification reaction until the amount of the nucleic acid having the variation in the nucleic acid amplification product reaches a threshold value, the variation is detected. In particular, in terms of the simplicity of implementation or the breadth of the applicable range, a method of detecting the variation based on the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification product reaches a threshold value is preferred.

[0250] In addition, accurate and highly sensitive detection can be performed by confirming the base sequence of the amplification product by direct sequencing, etc. However, as demonstrated in the examples described below, the method of the present invention can accurately and highly sensitively detect mutations without relying on sequence determination.

[0251] As methods for detecting mutations based on the total amount of nucleic acid amplification products, for example, there are a method in which the solution after the amplification reaction is deproteinized with a phenol-chloroform (1:1) solution, the aqueous layer is purified directly or after ethanol precipitation using an appropriate purification kit, and the absorbance of the purified solution at a wavelength of 260 nm is measured using an absorptiophotometer; a method in which the amplification product is developed by electrophoresis using agarose gel or polyacrylamide gel, and then detected by Southern hybridization using an appropriate probe; a method in which detection is performed by chromatographic hybridization using gold nanoparticles; a method in which the turbidity of a solution of the amplified nucleic acid is measured; a method in which the 5' end of the amplification primer is fluorescently labeled in advance, and after the amplification reaction, the fluorescence is collected on an imaging plate after electrophoresis using agarose gel or polyacrylamide gel, and detected using an appropriate detection device, etc.

[0252] As a method for detecting a mutation based on the number of amplification cycles of a nucleic acid amplification reaction until the total amount of nucleic acid amplification products reaches a threshold value, real-time PCR methods such as the intercalator method, the TaqManTM probe method, and the cycle probe method can be cited. Furthermore, the above threshold value can be automatically detected by a real-time PCR device (for example, Step One Plus Real-Time PCR System (manufactured by Thermo Fisher Scientific)).

[0253] The intercalator method is a method using an intercalator fluorescent dye (sometimes also referred to as an intercalator). An intercalator is a reagent that specifically binds to the base pairs of a double-stranded nucleic acid and fluoresces when irradiated with excitation light. The amount of the primer extension product can be known based on the detection of the fluorescence intensity derived from the intercalator. In the present embodiment, by real-time monitoring of this fluorescence intensity, the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification product reaches a threshold value can be known. In the present embodiment, any intercalator commonly used in this field can be used, for example, SYBRTM Green I (Molecular Probe Company), ethidium bromide, fluorene, etc. can be cited.

[0254] The TaqManTM probe method is a method for highly sensitive and quantitative detection of a trace amount of nucleic acid of a target by real-time PCR using an oligonucleotide probe that is hybridized to a specific region of a target nucleic acid and is labeled with a fluorescent group (reporter) at one end (usually, the 5' end) and the other end (usually, the 3' end) with an extinction group. The probe suppresses the fluorescence of the reporter by the extinction group under normal conditions. In a state where this fluorescent probe is completely hybridized with the detection region, PCR is performed using a DNA polymerase from its outside. An extension reaction by a DNA polymerase is performed, and the fluorescent probe is hydrolyzed by its exonuclease activity, and the reporter dye is freed and fluoresces. In this embodiment, by real-time monitoring of this fluorescence intensity, the number of amplification cycles of the nucleic acid amplification reaction until the total amount of nucleic acid amplification products reaches a threshold value can be known.

[0255] The cyclic probe method is a highly sensitive detection method that is a combination of a chimeric probe composed of RNA and DNA and RNase H. The probe is labeled with a fluorescent substance (reporter) on one side and a fluorescence extinguishing substance (quencher) on the other side. This probe does not fluoresce due to quenching in the Imtakt state, but after forming a hybrid with an amplification product complementary to the sequence, the RNA portion is cut off by RNase H, and it becomes strongly fluorescent. In this embodiment, the total amount of nucleic acid amplification products can be known by measuring this fluorescence intensity, and the number of amplification cycles of the nucleic acid amplification reaction until the total amount of nucleic acid amplification products reaches a threshold value can be known by real-time monitoring of the fluorescence intensity.

[0256] This method does not cause cleavage by RNase H when there is a mismatch near the RNA of the circulating probe, and enables very specific detection capable of identifying a one-base difference. The circulating probe can also be obtained by commissioning the design and synthesis of the circulating probe to an appropriate manufacturer.

[0257] Examples of methods for detecting mutations based on the amount of nucleic acid having base mutations in nucleic acid amplification products include Sanger sequencing and melting curve segmentation.

[0258] As a method for detecting a mutation based on the number of amplification cycles of the nucleic acid amplification reaction until the amount of nucleic acid having a base mutation in the nucleic acid amplification product reaches a threshold value, the above-mentioned real-time PCR method can be used using a probe that specifically binds to the nucleic acid having a base mutation.

[0259] [2. Kit for detecting mutations in a target base sequence]

[0260] In other embodiments, the present invention provides a kit for implementing the method for detecting a variation in the above-mentioned target base sequence. The kit according to this embodiment comprises:

[0261] A nucleic acid having a base sequence complementary to a standard sequence, containing at least one non-natural nucleotide residue, and

[0262] Diallylamine-sulfur dioxide copolymers containing the following constituent units:

[0263] The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0264]

Chemistry 23

[0265]

[0266] (Where R 1represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group), and

[0267] The structural unit (B) having a structure represented by the general formula (Ic):

[0268]

Chemistry 24

[0269]

[0270] In some cases, a primer set capable of amplifying a region containing a target base sequence by a nucleic acid amplification method, and

[0271] Other reagents used to carry out nucleic acid amplification reactions, if necessary.

[0272] The details of the nucleic acid, the diallylamine-sulfur dioxide copolymer, and the primer set are as described in the method for detecting a variation in a target base sequence, and the preferred embodiments and specific examples are also the same as those of the method.

[0273] As other reagents for implementing nucleic acid amplification reaction, nucleoside triphosphates, nucleic acid synthetase, and amplification reaction buffer can be mentioned. Nucleoside triphosphates can contain substrates (dNTP, rNTP, etc.) corresponding to nucleic acid synthetase. Nucleic acid synthetase is an enzyme corresponding to the nucleic acid amplification method as the object of the kit, and DNA polymerase, RNA polymerase, reverse transcriptase, etc. can be mentioned. As a buffer for amplification reaction, for example, Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good buffer, etc., which are used when implementing a common nucleic acid amplification reaction or hybridization reaction, can be mentioned, and its pH is also not particularly limited, preferably usually in the range of 5 to 9.

[0274] In addition, the kit according to this embodiment may contain reagents for detecting the above-mentioned nucleic acid amplification reaction product, for example, intercalators, fluorescently labeled probes, cycling probes, etc. used for real-time PCR.

[0275] In addition, other components generally used in nucleic acid amplification reaction reagents such as stabilizers and preservatives may be contained.

[0276] Furthermore, the primer set and other reagents for implementing the nucleic acid amplification reaction are necessary for implementing the nucleic acid amplification reaction, but may not be included in the kit. In addition, reagents for detecting nucleic acid amplification reaction products or other components such as stabilizers and preservatives are optional components in the nucleic acid amplification reaction and may not be included in the kit.

[0277] [3. Method for inhibiting amplification of a nucleic acid having a specified target base sequence in a nucleic acid amplification reaction]

[0278] Furthermore, in another embodiment, the present invention provides a method for inhibiting the amplification of a nucleic acid having a specified target base sequence. In this method, the following substances are used to perform a nucleic acid amplification reaction using the above nucleic acid as a template:

[0279] A nucleic acid having a base sequence complementary to a specified target base sequence, containing at least one non-natural nucleotide residue, and

[0280] Diallylamine-sulfur dioxide copolymers containing the following constituent units:

[0281] The structural unit (A) having a structure represented by the general formula (Ia) or (Ib) or a structure of an acid addition salt thereof:

[0282]

Chemistry 25

[0283]

[0284] (Where R 1 represents an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, which may have a hydroxyl group), and

[0285] The structural unit (B) having a structure represented by the general formula (Ic):

[0286]

Chemistry 26

[0287]

[0288] In the method of this embodiment, "sample" is a sample that is estimated to contain a nucleic acid that is a target of amplification inhibition, "target base sequence" is a base sequence that is a target of amplification inhibition, and "nucleic acid in the sample" is a nucleic acid that is likely to contain a base sequence that is a target of amplification inhibition. However, these terms do not mean that the "nucleic acid in the sample" used in this method actually contains a base sequence that is a target of amplification inhibition.

[0289] In addition, the "sandwich nucleic acid" in this embodiment has a base sequence complementary to the "target base sequence", and the presence of such a "sandwich nucleic acid" inhibits the amplification of the nucleic acid having the "target base sequence".

[0290] In this embodiment, by allowing the above-mentioned diallylamine·sulfur dioxide copolymer to coexist with a sandwich nucleic acid having a base sequence complementary to the target base sequence in the nucleic acid amplification reaction, the effect of inhibiting the amplification of the nucleic acid having the target base sequence of the sandwich nucleic acid can be enhanced, and when the nucleic acid having the target base sequence is present in a large amount in the sample, the amplification of the nucleic acid can be selectively inhibited.

[0291] The sample and other points of the nucleic acid in the sample, as described in the method for detecting mutations in the target base sequence, the preferred embodiments and specific examples are the same as those of the method. In addition, the details of the sandwich nucleic acid and the diallylamine-sulfur dioxide copolymer are also as described in the method for detecting mutations in the target base sequence, and the preferred embodiments and specific examples are the same as those of the method. In addition, the details of the implementation of the nucleic acid amplification reaction and the reagents for the nucleic acid amplification reaction are also as described in the method and kit for detecting mutations in the target base sequence, and the preferred embodiments and specific examples are the same as those of the method and kit. Of course, in the method of the present embodiment, it is not necessary to detect the amplification inhibition of the nucleic acid having the specified target base sequence, and the procedures and reagents necessary for such detection are implemented or used as needed.

[0292] [[4. Kit for Inhibiting Amplification of Nucleic Acid Having Specified Target Base Sequence in Nucleic Acid Amplification Reaction]]

[0293] Furthermore, in other embodiments, the present invention provides a kit for implementing a method for inhibiting the amplification of a nucleic acid having a target base sequence in the above nucleic acid amplification reaction. The kit according to the present embodiment includes:

[0294] A sandwich nucleic acid having a base sequence complementary to the target base sequence and containing at least one residue of a non-natural nucleotide, and

[0295] A diallylamine-sulfur dioxide copolymer containing the following structural units:

[0296] A structural unit (A) having a structure represented by the general formula (I-a) or (I-b) or a structure as its acid addition salt:

[0297] [[Chemical Formula 27]]

[0298]

[0299] (In the formula, R 1 represents an optionally hydroxy-substituted alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms), and

[0300] A structural unit (B) having a structure represented by the general formula (I-c):

[0301] [[Chemical Formula 28]]

[0302]

[0303] Optionally, a primer set capable of amplifying a region containing the target base sequence by a nucleic acid amplification method, and

[0304] Optionally, other reagents for implementing the nucleic acid amplification reaction.

[0305] The meanings of the terms "sample", "target base sequence", "nucleic acid in the sample", and "clamping nucleic acid" in this embodiment are the same as those described in the method for inhibiting the amplification of nucleic acids having the target base sequence in nucleic acid amplification reactions.

[0306] In addition, other aspects of the sample and the nucleic acid in the sample are the same as those described in the method for detecting mutations in the target base sequence, and the preferred embodiments and specific examples are also the same as those of the method. In addition, the details of the clamping nucleic acid and the diallylamine-sulfur dioxide copolymer are the same as those described in the method for detecting mutations in the target base sequence, and the preferred embodiments and specific examples are also the same as those of the method. In addition, the details of the implementation of nucleic acid amplification reactions and the reagents for implementing nucleic acid amplification reactions are the same as those described in the method and kit for detecting mutations in the target base sequence, and the preferred embodiments and specific examples are also the same as those of the method.

[0307] 【5. Nucleic Acid Amplification Inhibitor Enhancer for Enhancing Nucleic Acid Amplification Inhibition by Clamping Nucleic Acid】

[0308] Furthermore, in other embodiments, the present invention provides a nucleic acid amplification inhibitor enhancer that enhances the nucleic acid amplification inhibition effect of nucleic acids having a base sequence complementary to that of the clamping nucleic acid in nucleic acid amplification reactions.

[0309] This enhancer contains a diallylamine-sulfur dioxide copolymer, and the diallylamine-sulfur dioxide copolymer contains:

[0310] A structural unit (A) having a structure represented by the general formula (I-a) or (I-b) or a structure as its acid addition salt:

[0311]

Chemical Formula 29

[0312]

[0313] (In the formula, R 1 represents an optionally hydroxy group-bearing alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms), and

[0314] A structural unit (B) having a structure represented by the general formula (I-c):

[0315]

Chemical Formula 30

[0316]

[0317] The "nucleic acid amplification" performed using this enhancer is performed using a "sandwich nucleic acid having a base sequence complementary to the above-mentioned target base sequence and containing a non-natural nucleotide of at least 1 residue", and the terms "sample", "target base sequence", "nucleic acid in the sample" and "sandwich nucleic acid" in this embodiment have the same meaning as described in the method for inhibiting the amplification of a nucleic acid having a target base sequence in a nucleic acid amplification reaction.

[0318] In addition, the "nucleic acid amplification inhibition enhancer" according to this embodiment contains the above-mentioned diallylamine·sulfur dioxide copolymer, which is also described in the method for detecting a mutation in a target base sequence, and the preferred embodiments and specific examples are also the same as those of the method.

[0319] [Example]

[0320] Hereinafter, the present invention will be described more specifically by way of Examples, but the present invention is not limited thereto.

[0321] 【1. Effect of polymer addition on PCR reaction】

[0322] The diallylamine-sulfur dioxide copolymer having the above-mentioned structural unit (A) and structural unit (B) (Example) or a polymer having other structural units (Comparative Example) was added to the PCR reaction solution to confirm the influence on the PCR reaction.

[0323] [1-1. Aqueous polymer solution used]

[0324] The polymers shown in the following table were dissolved in water to prepare a 10 mass % polymer aqueous solution at pH 7.0. In the table, all product names are product names of NITTOBO MEDICAL Co., Ltd. The polymerization conditions of polymers 1 to 3 are as follows.

[0325] Polymer 1: 19.32 g of dilution water (amount to make a monomer concentration of 60% by mass) and 0.25 mol of diallylethylamine hydrochloride aqueous solution were added to a 100 ml three-necked flask equipped with a stirrer, a thermometer, and a glass stopper. After adding 0.25 mol of sulfur dioxide under cooling at 20° C. or less, a 28.5% by mass ammonium persulfate (APS) aqueous solution was added in 10 portions so that the amount of ammonium persulfate in the aqueous solution was 1.5% by mass relative to the total amount of monomers, and polymerization was carried out at 15 to 30° C.

[0326] Polymer 2: 4.29 g of dilution water (amount to make monomer concentration 70% by mass), 0.13 mol of diallylpropylamine hydrochloride aqueous solution and 35% by mass hydrochloric acid were added to 0.27 g of a 100 ml three-necked flask equipped with a stirrer, a thermometer and a glass stopper. After adding 0.13 mol of sulfur dioxide under cooling at 20° C. or less, a 28.5% by mass ammonium persulfate (APS) aqueous solution was added in 10 portions so that the amount of ammonium persulfate was 1.5% by mass relative to the total amount of monomers, and polymerization was carried out at 15 to 30° C.

[0327] Polymer 3: 248.74 g (1.0 mol) of 65.0% diallyldimethylammonium chloride, 49.03 g (0.5 mol) of anhydrous maleic acid, 89.77 g of distilled water, and sulfur dioxide were added to a 1L four-mouth flask equipped with a stirrer, a thermometer, and a cooling tube, and cooled to 20°C. A 28.5% by mass ammonium persulfate (APS) aqueous solution was added in 9 portions so that the amount of ammonium persulfate was 1.5% by mass relative to the total amount of monomers. The internal temperature was raised to 60°C, and the above-mentioned ammonium persulfate (APS) aqueous solution was added in 5 portions so that the amount of ammonium persulfate was 5.0% by mass relative to the total amount of monomers, and the reaction was continued for 72 hours.

[0328]

Table 1

[0329]

[0330] (*) In Comparative Examples 1 to 3, the diallylamine unit does not correspond to the constituent unit (A), but is recorded in the column of the constituent unit (A) for the convenience of explanation in this table. In addition, the polymer of Comparative Example 3 is composed of three constituent units of diallyldimethylammonium chloride, sulfur dioxide, and maleic acid, and the molar ratio of each constituent unit is 2:1:1.

[0331] 【1-2. PCR reaction solution】

[0332] A PCR reaction solution having the composition shown in the following table was prepared.

[0333]

Table 2

[0334] Element content <![CDATA[2×PCR Buffer TB Green Premix Dimer Eraser *1 (Made by Takara Biotech)]]> 10.0μL 10 μM forward primer (synthesized on behalf of Fasmac) 0.6μL 10 μM reverse primer (synthesized on behalf of Fasmac) 0.6μL 50×ROX dye (produced by Takara Bio) 0.4μL 50ng / μL template DNA 1.0μL Polymer aqueous solution 2.0μL Nuclease-free water (Thermo Fisher Scientific) 5.4μL

[0335] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ The final concentration of each polymer aqueous solution in the PCR reaction solution was 0.01% by mass.

[0336] 【1-3. Primers and template DNA】

[0337] The sequences of the forward primer and reverse primer used in the PCR reaction are shown in the following table.

[0338]

Table 3

[0339] Primers sequence Sequence number Forward Primer GCCTGCTGAA AATGACTGAA TATA SEQ ID NO: 1 Reverse primer CAAGATTTAC CTCTATTGTT GGA SEQ ID No: 2

[0340] Each primer was designed using Primer3 (available from http: / / frodo.wi.mit.edu) with the KRAS gene (the base sequence can be confirmed at, for example, NCBI (DB name)) as a target.

[0341] As template DNA, a genome extracted from HCC70 cells containing the following base sequence (sequence to be amplified) was used.

[0342]

Chemistry 31

[0343]

[0344] Furthermore, the HCC70 cell genome contains the above-mentioned base sequence, and the base sequence adjacent thereto is as follows.

[0345]

Chemistry 32

[0346]

[0347] 【1-4. Real-time PCR system】

[0348] Step One Plus Real-Time PCR System (manufactured by Thermo Fisher Scientific) was used.

[0349] 【1-5. PCR reaction】

[0350] 20 μL of the prepared PCR reaction solution was placed in a real-time PCR system, and step (i) described in the following table was performed, and then steps (ii) to (iv) were repeated for 40 cycles.

[0351]

Table 4

[0352] step temperature time i 95℃ 30 seconds ii 95℃ 5 seconds iii 55℃ 30 seconds iv 72℃ 30 seconds V 95℃ 15 seconds vi 60℃ 60 seconds vii 95℃ 15 seconds

[0353] After 40 cycles, proceed to steps (v) to (vii) to terminate the reaction. The fluorescence intensity of the reaction solution is measured in each cycle to monitor the amount of DNA amplification. In addition, after the reaction is completed, the presence or absence of inhibition of the PCR reaction is determined by whether the fluorescence intensity reaches the threshold line. The threshold line is automatically calculated by the above device. Sometimes, when it is necessary to set it lower than usual according to the experiment, it is manually set to a ΔRn value between 0.7 and 1.2 in order to compare with other experiments in the same column.

[0354] 【1-5. Results】

[0355] The success or failure of the PCR reaction is shown in the following table.

[0356]

Table 5

[0357]

[0358]

[0359] ○: Does not inhibit PCR reaction.

[0360] ×: PCR reaction is inhibited.

[0361] In addition, the nucleic acid amplification curve when the polymer of Example 1 (diallylmethylamine hydrochloride·sulfur dioxide copolymer) was added as an example of the nucleic acid amplification curve when the PCR reaction was not inhibited and the nucleic acid amplification curve when the comparative example 1 (diallylamine hydrochloride·sulfur dioxide copolymer) was added as an example of the nucleic acid amplification curve when the PCR reaction was inhibited are shown in FIG. Figure 1 .

[0362] As described above, when the polymers of Examples 1 to 3, which are copolymers of diallylamine and sulfur dioxide containing cationic structural units containing a tertiary amine in the structure and structural units derived from a sulfur dioxide monomer, were added, the PCR reaction was not inhibited. On the other hand, when the polymers of Comparative Examples 1 to 3, which are copolymers containing cationic structural units containing a secondary or quaternary amine and structural units derived from a sulfur dioxide monomer or sulfur dioxide and maleic acid, were added, the PCR reaction was inhibited.

[0363] 【2. Enhancement of the selective amplification inhibition effect of wild-type genes clamped by BNA by polymers or monomers】

[0364] Evaluation of the Enhanced Effect of Inhibiting the Amplification of the Wild-Type KRAS Gene in BNA Clamp PCR In Test 1, it was confirmed that the polymers of Examples 1 to 3 and the monomer (diallylmethylamine hydrochloride) from which some of their constituent units were derived did not inhibit the PCR reaction.

[0365] 【2-1. Polymers or monomers used】

[0366] In this test, the polymers of Examples 1 to 3 and diallylmethylamine hydrochloride (Comparative Example 4) were used. Each polymer or monomer was dissolved in water to prepare a 0.25 mass % polymer aqueous solution or monomer aqueous solution at pH 7.0.

[0367] 【2-2. Kit used】

[0368] Use the accessories of the BNA Clamp KRAS Enrichment Kit (RIKEN Genesis) as a kit for detecting mutant KRAS genes. The following details will be described. Primer set and BNA clamp use the kit accessories. In addition, the reaction conditions for real-time PCR follow the kit protocol.

[0369] [[2-3. Primers, BNA Clamp, and Template DNA]]

[0370] Use the forward primer, reverse primer, and BNA clamp shown in the following table.

[0371] [[Table 6]]

[0372] sequence Sequence number Forward primer CTGAATATAA ACTTGTGGTA GTTGG SEQ ID NO: 5 Reverse primer GTCCTGCACC AGTAATATGC SEQ ID NO: 6 BNA Clip C*C*T*AC*GC*C*AC*C* SEQ ID NO: 7

[0373] *: Represents the base of an artificial nucleotide.

[0374] In addition, as the template DNA with the standard base sequence, use the genome of HCC70 cells with the wild-type KRAS gene containing the following base sequence (amplification target sequence; bases different from the base sequence of SEQ ID NO: 10 described later are underlined and bolded).

[0375] [[Chemical 33]]

[0376]

[0377] Furthermore, in the HCC70 cell genome, the above base sequence and the adjacent base sequence are as follows (bases different from the base sequence of SEQ ID NO: 11 described later are underlined and bolded).

[0378] [[Chemical 34]]

[0379]

[0380] On the other hand, as the template DNA with a mutation relative to the standard base sequence, use the genome of MDA-MB-231 cells with the mutant KRAS gene containing the following base sequence (amplification target sequence; bases different from the base sequence of SEQ ID NO: 8 described above are underlined and bolded).

[0381] [[Chemical 35]]

[0382]

[0383] Furthermore, in the MDA-MB-231 cell genome, the above base sequence and the adjacent base sequence are as follows (bases different from the base sequence of SEQ ID NO: 9 described above are underlined and bolded).

[0384]

Chemical 36

[0385]

[0386] 【2-4.BNA Clamp PCR Reaction Solution】

[0387] Prepare Reaction Solutions 1 to 4 with the compositions shown in the following table. In Reaction Solution 1 or Reaction Solution 2, the concentration of the polymer or monomer is 0.025% by mass.

[0388]

Table 7: Reaction Solution 1 (Polymer or Monomer + , BNA Clamp + )

[0389]

[0390] *1 : Containing Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0391]

Table 8: Reaction Solution 2 (Polymer or Monomer + , BNA Clamp - )

[0392]

[0393]

[0394] *1 : Containing Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0395]

Table 9: Reaction Solution 3 (Polymer or Monomer - , BNA Clamp + )

[0396]

[0397] *1 : Containing Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0398]

Table 10: Reaction Solution 4 (Polymer or Monomer - , BNA Clamp - )

[0399]

[0400] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0401] 【2-5. Real-time PCR system】

[0402] The Step One Plus Real-Time PCR System (Thermo Fisher Scientific) was used.

[0403] 【2-6. PCR reaction】

[0404] 20 μL of each reaction solution was weighed into a PCR tube, and the PCR tube was placed in a real-time PCR system. After performing step (i) described in the following table, steps (ii) to (iv) were repeated for 50 cycles. After 50 cycles, steps (v) to (vii) were performed to terminate the reaction. The fluorescence intensity of the reaction solution was measured in each cycle to monitor the amount of DNA amplification and obtain the proliferation curve of each reaction solution.

[0405]

Table 11

[0406] step temperature time i 95℃ 30 seconds ii 95℃ 5 seconds iii 58℃ 30 seconds iv 72℃ 30 seconds V 95℃ 15 seconds vi 60℃ 60 seconds vii 95℃ 15 seconds

[0407] [2-7. Calculation of ΔCt(WT) and ΔCt(Variant) when a polymer is added or not added, calculation of ΔΔCt when a polymer is added or not added, and calculation of ΔΔΔCt]

[0408] The above-mentioned PCR reaction was carried out using reaction solution 1 (polymer added, BNA clamp added), reaction solution 2 (polymer added, BNA clamp not added), reaction solution 3 (polymer not added, BNA clamp added) and reaction solution 4 (polymer not added, BNA clamp not added) as template DNA, and the wild-type KRAS gene or the mutant KRAS gene was used to obtain an amplification curve, and the respective Ct values ​​were obtained from the amplification curve of each condition according to the set threshold line (in most experimental distinctions, the threshold line is automatically calculated by the device. Sometimes, when it is necessary to set it lower than usual according to the experiment, in order to compare with other experiments in the same column, the ΔRn value is manually set to between 0.7 and 1.2).

[0409] Next, ΔCt(WT) and ΔCt(variant) in the case where no polymer was added were calculated from the following formula.

[0410]

Number 1

[0411] ΔCt(WT) when no polymer was added = Ct(WT) when reaction solution 3 (no polymer was added, BNA clamp was added) - Ct(WT) when reaction solution 4 (no polymer was added, BNA clamp was not added)

[0412]

Number 2

[0413] ΔCt (variant) when no polymer was added = Ct (variant) when reaction solution 3 (no polymer was added, BNA clamp was added) - Ct (variant) when reaction solution 4 (no polymer was added, BNA clamp was not added)

[0414] Furthermore, in order to confirm non-specific amplification in the intercalator-free method, ΔΔCt in the case where no polymer was added was calculated according to the following formula.

[0415]

Number 3

[0416] ΔΔCt when no polymer is added = ΔCt when no polymer is added (WT) - ΔCt when no polymer is added (variant)

[0417] When ΔΔCt>0 in the case where no polymer was added, it was understood that it was nonspecific amplification in the non-intercalator method.

[0418] Next, ΔCt(WT) and ΔCt(variant) in the case of adding a polymer were calculated from the following formulas. Furthermore, ΔΔCt in the case of adding a polymer was calculated in order to confirm non-specific amplification in the intercalator-free method.

[0419]

Number 4

[0420] ΔCt(WT) when polymer was added = Ct(WT) when reaction solution 1 (polymer added, BNA clamp added) - Ct(WT) when reaction solution 2 (polymer added, BNA clamp not added)

[0421]

Number 5

[0422] ΔCt (variant) in the case of polymer addition = Ct (variant) in the case of reaction solution 1 (polymer addition, BNA clamp addition) - Ct (variant) in the case of reaction solution 2 (polymer addition, BNA clamp not added)

[0423]

Number 6

[0424] ΔΔCt of the case where the polymer was added = ΔCt of the case where the polymer was added (WT) - ΔCt of the case where the polymer was added (variant)

[0425] When ΔΔCt>0 in the case of adding a polymer, it is understood that it is nonspecific amplification in the method without an intercalator.

[0426] Finally, ΔΔΔCt was calculated from the following formula as an index for evaluating the enhancement of the effect of selectively suppressing the amplification of the wild-type gene clamped by the BNA by the addition of the polymer.

[0427]

Number 7

[0428] ΔΔΔCt = ΔΔCt when polymer was added - ΔΔCt when polymer was not added

[0429] Even considering the fluctuation of the results that may occur in the experiment, when using a variation detection method based on the number of amplification cycles of the wild-type gene as a benchmark like the intercalator method, when ΔΔΔCt≥1.00, it can be evaluated that the selective amplification inhibitory effect of the wild-type gene clamped by the BNA is fully enhanced. In addition, when using a variation detection method based on the number of amplification cycles of the wild-type gene as a benchmark like the intercalator method, when ΔΔΔCt≥4.00, it can be evaluated that the selective amplification inhibitory effect of the wild-type gene clamped by the BNA is significantly enhanced. Furthermore, when using a variation detection method based on the number of amplification cycles of the wild-type gene as a benchmark like the intercalator method, when ΔΔΔCt≥8.00, it can be evaluated that the selective amplification inhibitory effect of the wild-type gene clamped by the BNA is extremely significantly enhanced. Furthermore, ΔΔΔCt=3.20 is the benchmark for an approximately 10-fold increase in sensitivity.

[0430] 【2-8. Results】

[0431] Next, the test results are comprehensively displayed.

[0432]

Table 12

[0433] Polymer or monomer ΔΔΔCt Example 1 Diallylmethylamine hydrochloride·sulfur dioxide copolymer 11.74 Example 2 Diallylethylamine hydrochloride·sulfur dioxide copolymer 6.05 Example 3 Diallylpropylamine hydrochloride·sulfur dioxide copolymer 2.91 Comparative Example 4 Diallylmethylamine hydrochloride 0.95

[0434] The above test results show that the addition of the polymers of Examples 1 to 3 does not inhibit the amplification of the mutant gene, but enhances the selective amplification inhibition effect of the wild-type gene sandwiched by the BNA.

[0435] On the other hand, although the monomer of Comparative Example 4 also enhanced the selective amplification inhibitory effect of the wild-type gene clamped by BNA, a sufficient inhibitory enhancement effect as that of the polymers of Examples 1 to 3 was not confirmed.

[0436] In addition, it was found that when adding monomers, the error (deviation of results) was large and the effect was unstable. For this error (deviation of results), since monomers can generate free radicals by heat, light, etc., the possibility of free radical polymerization is considered. From this experiment, it was found that the selective amplification inhibition effect of wild-type genes clamped by BNA was enhanced more reproducibly, which was more advantageous than the monomer-polymer structure.

[0437] 【3. Higher sensitivity of PCR by BNA-addition to polymer】

[0438] Using the polymer of Example 1, which was confirmed in the above-mentioned test to have an effect of enhancing the selective amplification inhibition effect of the wild-type gene by the BNA clamp, an experiment was conducted to increase the sensitivity of the BNA clamp PCR.

[0439] 【3-1. Use of polymer】

[0440] The polymer of Example 1 was dissolved in water to prepare a 10% by mass aqueous polymer solution at pH 7.0. The obtained 10% by mass aqueous polymer solution was further diluted with water to prepare a 0.1% by mass aqueous polymer solution, which was used in the preparation of the reaction solution.

[0441] 【3-2. Kit used】

[0442] The same accessories of the kit as in Experiment 2 were used.

[0443] 【3-3. Template DNA】

[0444] As the template DNA having the standard base sequence, the genome of HCC70 cells having the wild-type KRAS gene having the base sequence of SEQ ID NO: 8 was used. In addition, as the template DNA having a mutation relative to the standard base sequence, the genome of MDA-MB-231 cells having the mutant KRAS gene having the base sequence of SEQ ID NO: 10 was used.

[0445] In this experiment, the total amount of template DNA was adjusted to 50 ng in 2.0 μL, and the mass % of template DNA of the mutant gene in the total template DNA was adjusted to be as shown in the table below. The template DNA of the wild-type gene and the template DNA of the mutant gene were mixed, and the resulting template DNA mixtures and the template DNA of the wild-type gene were used in the PCR reaction.

[0446]

Table 13

[0447] Variant ratio Variant copy number 10% variant (5ng) <![CDATA[1.65×10 3 Number of copies]]> 1% variant (0.5ng) <![CDATA[1.65×10 2 Number of copies]]> 0.1% variant (0.05ng) 1.65×10 copies 0.01% variant (0.005ng) 1.65 copies

[0448] 【3-4. Primers and BNA clamp】

[0449] The same primers and BNA clamps as in Experiment 2 were used.

[0450] 【3-5. BNA clamp PCR reaction solution】

[0451] Reaction solutions 1 and 2 having the compositions shown in the following table were prepared. In the reaction solution 1, the polymer concentration was 0.01% by mass.

[0452] [Table 14: Reaction solution 1 (polymer + 、BNA clip+ )】

[0453] Element content <![CDATA[PCR Buffer TB Green Premix Dimer Eraser *1 (Made by Takara Biotech)]]> 10.0μL 10 μM forward primer (Riken Genesis) 0.6μL 10 μM reverse primer (Riken Genesis) 0.6μL ROX dye (produced by Takara Bio) 0.4μL 10 μM BNA clamp (Riken Genesis) 0.2μL <![CDATA[50 ng / μL template DNA (WT *2 or WT and variants *3 mixture *4 )]]> 2.0μL 0.1 mass% polymer aqueous solution 2.0μL Nuclease-free water (Thermo Fisher Scientific) 4.2μL

[0454] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0455] *2 :Genomic DNA of HCC70 cells

[0456] *3 : Genomic DNA of MDA-MB-231 cells

[0457] *4 : The entire mixture contains 50 ng / μL of template DNA in 2.0 μL.

[0458] [Table 15: Reaction solution 2 (polymer-, BNA clamp +)]

[0459] Element content <![CDATA[PCR Buffer TB Green Premix Dimer Eraser *1 (Made by Takara Biotech)]]> 10.0μL 10 μM forward primer (Riken Genesis) 0.6μL 10 μM reverse primer (Riken Genesis) 0.6μL ROX dye (produced by Takara Bio) 0.4μL 10 μM BNA clamp (Riken Genesis) 0.2μL <![CDATA[50 ng / μL template DNA (WT *2 or WT and variants *3 mixture *4 )]]> 2.0μL Polymer aqueous solution 0μL Nuclease-free water (Thermo Fisher Scientific) <![CDATA[6.2μL *2 ]]>

[0460] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0461] *2 :Genomic DNA of HCC70 cells

[0462] *3 : Genomic DNA of MDA-MB-231 cells

[0463] *4 : The entire mixture contains 50 ng / μL of template DNA in 2.0 μL.

[0464] 【3-6. Real-time PCR system and PCR reaction】

[0465] The PCR reaction was carried out under the same conditions as in Experiment 2 using the same real-time PCR system as in Experiment 2.

[0466] [3-7. Determination of δCt (variant 10% to 0.01%) when a polymer is added or not added, and calculation of δδCt (variant 10% to 0.01%) when a polymer is added or not added]

[0467] Reaction solution 1 (polymer added, BNA clamp added) and reaction solution 2 (polymer not added, BNA clamp added) were subjected to the above-mentioned PCR reaction using template DNA or a mixture of each template DNA to obtain an amplification curve. Ct (variant 10% to 0%) was determined from the amplification curve of each condition in the same manner as in Experiment 2. As shown in the table below, the Ct (variant 10% to 0.01%) of each mutant gene template concentration was subtracted from Ct (variant 0%, WT), and each δCt (variant 10% to 0.01%) was calculated for the case with or without polymer addition.

[0468] [Table 16: Calculation formula of δCt (variant 10% to 0.01%) when no polymer is added]

[0469] Variants 10％ 1％ 0.10％ 0.01％ 0％ ct ① ② ③ ④ ⑤ δct ⑤-① ⑤-② ⑤-③ ⑤-④

[0470] [Table 17: Calculation formula of δCt (variant 10% to 0.01%) when polymer is added]

[0471] Variants 10％ 1％ 0.10％ 0.01％ 0％ Ct A B C D E δCt EA EB EC ED

[0472] Finally, δCt (variant 10% to 0.01%) when no polymer was added was subtracted from δCt (variant 10% to 0.01%) when the polymer was added, δδCt (variant 10% to 0.01%).

[0473] When δδCt>0, it means that the mutant gene is specifically detected by adding the polymer, and the amplification curve of the mutant gene is distinguished from the amplification curve of the wild-type gene (reference: Patent Document 11).

[0474] 【3-8. Results】

[0475] The test results are compared with the following table and Figure 2 Comprehensive display.

[0476]

Table 18

[0477] Variant ratio δCt without polymer addition δCt of the case of polymer addition δδCt 10% variant 7.88 10.68 2.80 1% variant 4.85 7.00 2.15 0.1% variant 2.67 4.62 1.95 0.01% variant -0.68 3.81 4.49

[0478] The results of this experiment showed that when the polymer was not added, the variant ratio was 0.01% by mass, resulting in δCt less than 0, and the amplification curve of the mutant gene could not be distinguished from the amplification curve of the wild-type gene. When the polymer of Example 1 was added, the amplification curve of the mutant gene could be distinguished from the amplification curve of the wild-type gene even at a variant ratio of 0.01%. In addition, although the experiment was not conducted on the reaction solution without the addition of the BNA clamp, the results of Experiment 2 showed that in the reaction solution without the addition of the BNA clamp, no nonspecific amplification occurred regardless of the presence or absence of the addition of the polymer.

[0479] 【4. Adaptability to Mutant Genes Other than KRAS 1 (Enhancement by Polymer of Selective Amplification Inhibition Effect of Wild-Type Gene by BNA Clamp)】

[0480] 【4-1. Polymers Used】

[0481] The polymer of Example 1 was dissolved in water to prepare a 0.1% by mass polymer solution, which was used in the preparation of the reaction solution.

[0482] 【4-2. Kits Used】

[0483] The BNA Clamp BRAF Enrichment Kit (RIKEN Genesis) was used as a kit for detecting mutant BRAF genes. The primer set and BNA clamp were used as accessories in the kit. In addition, the reaction conditions for real-time PCR were based on the kit protocol.

[0484] 【4-3. Template DNA】

[0485] As the template DNA with the standard base sequence, the genomic DNA of HCC70 cells with the wild-type BRAF gene having the following base sequence (the bases different from the base sequence of SEQ ID NO: 13 described later are underlined and bolded) was used.

[0486]

Chemical 37

[0487]

[0488] Furthermore, it should be noted that the above sequence contains base sequences other than the amplification target sequence.

[0489] On the other hand, as the template DNA with a mutation relative to the standard base sequence, the genomic DNA of DU4475 cells with the mutant BRAF gene having the following base sequence (the bases different from the base sequence of SEQ ID NO: 12 described above are underlined and bolded) was used.

[0490]

Chemical 38

[0491]

[0492] Furthermore, it should be noted that the above sequence contains base sequences other than the amplification target sequence.

[0493] 【4-4. BNA Clamp PCR Reaction Solution】

[0494] Reaction solutions 1 to 4 with the compositions shown in the following table were prepared. Furthermore, the polymer concentration in reaction solutions 1 and 2 was 0.01% by mass.

[0495] [Table 19: Reaction solution 1 (polymer + 、BNA clip + )】

[0496]

[0497] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0498] [Table 20: Reaction solution 2 (polymer + 、BNA clip - )】

[0499]

[0500]

[0501] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0502] [Table 21: Reaction solution 3 (polymer - 、BNA clip + )】

[0503]

[0504] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0505] [Table 22: Reaction solution 4 (polymer - 、BNA clip - )】

[0506]

[0507] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0508] 【4-5. Real-time PCR system and PCR reaction】

[0509] The PCR reaction was carried out in the same manner as in Experiment 2 using the same system as in Experiment 2.

[0510] [4-6. Calculation of ΔCt(WT) and ΔCt(Variant) when a polymer is added or not added, calculation of ΔΔCt when a polymer is added or not added, and calculation of ΔΔΔCt]

[0511] The same procedure as in Test 2 was carried out.

[0512] 【4-7. Results】

[0513] Next, the test results are comprehensively displayed.

[0514]

Table 23

[0515] Polymer or monomer ΔΔΔCt Example 1 Diallylmethylamine hydrochloride·sulfur dioxide copolymer 7.76

[0516] As described above, the addition of the polymer of Example 1 significantly enhanced the selective amplification inhibitory effect of the wild-type BRAF gene sandwiched by BNA, similarly to the KRAS gene.

[0517] [5. Possibility of adapting to mutant genes other than KRAS 2 (Increased sensitivity of PCR by polymer-added BNA)]

[0518] 【5-1. Polymers used】

[0519] The polymer of Example 1 was dissolved in water to prepare a 0.1 mass % polymer solution, which was used in the preparation of the reaction solution.

[0520] 【5-2. Kit used】

[0521] The same kit as in Experiment 4 was used.

[0522] 【5-3. Template DNA】

[0523] The same template DNA as in Experiment 4 was used.

[0524] In this experiment, the total amount of template DNA was adjusted to 50 ng in 2.0 μL, and the mass % of template DNA of the mutant gene in the total template DNA was adjusted to be as shown in the table below. The template DNA of the wild-type gene and the template DNA of the mutant gene were mixed, and the resulting template DNA mixtures and the template DNA of the wild-type gene were used in the PCR reaction.

[0525]

Table 24

[0526] Variant ratio Variant copy number 10% variant (5ng) <![CDATA[1.65×10 3 Number of copies]]> 1% variant (0.5ng) <![CDATA[1.65×10 2 Number of copies]]> 0.1% variant (0.05ng) 1.65×10 copies 0.01% variant (0.005ng) 1.65 copies

[0527] 【5-4. Primers and BNA clamp】

[0528] The same primers and BNA clamps as in Experiment 4 were used.

[0529] 5-5. BNA clamp PCR reaction solution

[0530] Reaction solutions 1 to 4 having the compositions shown in the following table were prepared. The polymer concentration in the reaction solution 1 was 0.01% by mass.

[0531] [Table 25: Reaction solution 1 (polymer + 、BNA clip + )】

[0532] Element content <![CDATA[PCR Buffer TB Green Premix Dimer Eraser *1 (Made by Takara Biotech)]]> 10.0μL 10 μM forward primer (Riken Genesis) 0.6μL 10 μM reverse primer (Riken Genesis) 0.6μL ROX dye (produced by Takara Bio) 0.4μL 10 μM BNA clamp (Riken Genesis) 0.2μL <![CDATA[50 ng / μL template DNA (WT *2 or WT and variants *3 mixture *4 )]]> 2.0μL 0.1 mass% polymer aqueous solution 2.0μL Nuclease-free water (Thermo Fisher Scientific) 4.2μL

[0533] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2 + and fluorescent dye (TB Green).

[0534] *2 :Genomic DNA of HCC70 cells

[0535] *3 : Genomic DNA of DU4475 cells

[0536] *4 : The entire mixture contains 50 ng / μL of template DNA in 2.0 μL.

[0537] [Table 26: Reaction solution 2 (polymer - 、BNA clip + )】

[0538] Element content <![CDATA[PCR Buffer TB Green Premix Dimer Eraser *1 (Made by Takara Biotech)]]> 10.0μL 10 μM forward primer (Riken Genesis) 0.6μL 10 μM reverse primer (Riken Genesis) 0.6μL ROX dye (produced by Takara Bio) 0.4μL 10 μM BNA clamp (Riken Genesis) 0.2μL <![CDATA[50 ng / μL template DNA (WT *2 or WT and variants *3 mixture *4 )]]> 2.0μL Polymer aqueous solution 0μL Nuclease-free water (Thermo Fisher Scientific) 6.2μL

[0539] *1 :Contains Tag polymerase (TaKaRa Ex Taq HS), dNTP mixture, Mg 2+ and fluorescent dye (TB Green).

[0540] *2 :Genomic DNA of HCC70 cells

[0541] *3 : Genomic DNA of DU4475 cells

[0542] *4 : The entire mixture contains 50 ng / μL of template DNA in 2.0 μL.

[0543] 【5-6. Real-time PCR system and PCR reaction】

[0544] The PCR reaction was carried out in the same manner as in Experiment 2 using the same apparatus as in Experiment 2.

[0545] [5-7. Determination of δCt (variant 10% to 0.01%) when a polymer is added or not added, and calculation of δδCt (variant 10% to 0.01%) when a polymer is added or not added]

[0546] The same procedure as in Test 3 was carried out.

[0547] 【5-8. Results】

[0548] The test results are compared with the following table and Figure 3 Comprehensive display.

[0549]

Table 27

[0550] Variant ratio δCt without polymer addition δCt of the case of polymer addition δδCt 10% variant 10.76 14.87 4.11 1% variant 6.81 11.33 4.52 0.1% variant 4.60 7.74 3.14 0.01% variant -3.75 2.96 6.71

[0551] The results of this experiment show that when no polymer is added, the variant ratio is 0.01%, resulting in a δCt of less than 0, and the amplification curve of the mutant gene cannot be distinguished from the amplification curve of the wild-type gene. When the polymer of Example 1 is added, the amplification curve of the mutant gene can be distinguished from the amplification curve of the wild-type gene even at a variant ratio of 0.01%.

Claims

1. A method for detecting a variation in a target base sequence of a nucleic acid in a sample relative to a reference base sequence for a non-diagnostic purpose, wherein: The following substances are used to carry out a nucleic acid amplification reaction using the nucleic acid in the sample as a template: A nucleic acid comprising a base sequence complementary to the standard base sequence, comprising a non-natural nucleotide of at least one residue, and Diallylamine-sulfur dioxide copolymers containing the following constituent units: The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof: In the formula, R 1 represents an alkyl group having 1 to 10 carbon atoms, and The structural unit (B) having a structure represented by the general formula (Ic): The presence of the variation is determined based on the total amount of nucleic acid amplification products obtained by the nucleic acid amplification reaction, the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification products reaches a threshold value, the amount of nucleic acid having the variation in the nucleic acid amplification products, or the number of amplification cycles of the nucleic acid amplification reaction until the amount of nucleic acid having the variation in the nucleic acid amplification products reaches a threshold value.

2. The method according to claim 1, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

3. The method according to claim 1, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

4. The method according to any one of claims 1 to 3, wherein the non-natural nucleotide is BNA.

5. The method according to any one of claims 1 to 3, wherein the nucleic acid amplification is performed by real-time PCR.

6. The method according to any one of claims 1 to 3, wherein the nucleic acid in the sample is genomic DNA.

7. The method according to any one of claims 1 to 3, wherein when the nucleic acid amplification reaction is performed, a nucleic acid amplification reaction using the nucleic acid having the standard base sequence as a template is also performed. When the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification product reaches a threshold value when the nucleic acid in the sample is used as a template is smaller than the number of amplification cycles of the nucleic acid amplification reaction until the total amount of the nucleic acid amplification product reaches a threshold value when the nucleic acid having the standard base sequence is used as a template, it is determined that the variation exists in the target base sequence of the nucleic acid in the sample.

8. Use of the following substances in the manufacture of a kit for detecting a variation in a target base sequence of a nucleic acid in a sample relative to a standard base sequence: A nucleic acid comprising a base sequence complementary to the standard base sequence, comprising at least one non-natural nucleotide residue, and Diallylamine-sulfur dioxide copolymers containing the following constituent units: The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof: In the formula, R 1 represents an alkyl group having 1 to 10 carbon atoms, and The structural unit (B) having a structure represented by the general formula (Ic):

9. The use according to claim 8, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer, or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

10. The use according to claim 8, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

11. The use according to any one of claims 8 to 10, wherein the non-natural nucleotide is BNA.

12. The use according to any one of claims 8 to 10, wherein the nucleic acid in the sample is genomic DNA.

13. A kit for detecting a variation in a target base sequence of a nucleic acid in a sample relative to a standard base sequence, comprising: A nucleic acid having a base sequence complementary to the standard base sequence, containing at least one non-natural nucleotide residue, and Diallylamine-sulfur dioxide copolymers containing the following constituent units: The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof: In the formula, R 1 represents an alkyl group having 1 to 10 carbon atoms, and The structural unit (B) having a structure represented by the general formula (Ic):

14. The kit according to claim 13, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

15. The kit according to claim 13, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

16. The kit according to any one of claims 13 to 15, wherein the non-natural nucleotide is BNA.

17. A method for inhibiting amplification of a nucleic acid in a sample having a specified target base sequence in a nucleic acid amplification reaction, wherein: The following substances were used to carry out a nucleic acid amplification reaction using the nucleic acid as a template: A nucleic acid having a base sequence complementary to the target base sequence, containing at least one non-natural nucleotide residue, and Diallylamine-sulfur dioxide copolymer containing the following constituent units: The structural unit (A) having a structure represented by the general formula (Ia) or (Ib) or a structure of an acid addition salt thereof: In the formula, R 1 represents an alkyl group having 1 to 10 carbon atoms, and The structural unit (B) having a structure represented by the general formula (Ic):

18. The method of claim 17, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

19. The method according to claim 17, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

20. The method of any one of claims 17 to 19, wherein the non-natural nucleotide is a BNA.

21. A kit for inhibiting amplification of a nucleic acid having a target base sequence in a nucleic acid amplification reaction, comprising: A nucleic acid having a base sequence complementary to the target base sequence, containing at least one non-natural nucleotide residue, and Diallylamine-sulfur dioxide copolymer containing the following constituent units: The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof: In the formula, R 1 represents an alkyl group having 1 to 10 carbon atoms, and The structural unit (B) having a structure represented by the general formula (Ic):

22. The kit according to claim 21, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

23. The kit according to claim 21, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

24. The kit according to any one of claims 21 to 23, wherein the non-natural nucleotide is BNA.

25. A nucleic acid amplification inhibition enhancer for enhancing the effect of nucleic acid amplification inhibition by sandwich nucleic acid, comprising a diallylamine-sulfur dioxide copolymer, wherein the diallylamine-sulfur dioxide copolymer comprises the following constituent units: The structural unit (A) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof: In the formula, R 1 represents an alkyl group having 1 to 10 carbon atoms, and The structural unit (B) having a structure represented by the general formula (Ic):

26. The nucleic acid amplification inhibition enhancer according to claim 25, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer, a diallylmethylamine acid addition salt·sulfur dioxide copolymer, a diallylethylamine·sulfur dioxide copolymer or a diallylethylamine acid addition salt·sulfur dioxide copolymer.

27. The nucleic acid amplification inhibition enhancer according to claim 25, wherein the diallylamine·sulfur dioxide copolymer is a diallylmethylamine·sulfur dioxide copolymer or a diallylmethylamine acid addition salt·sulfur dioxide copolymer.

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