Agent for increasing the melting temperature of double-stranded nucleic acid and its use

Abstract

The present invention provides a novel agent for increasing the melting temperature (Tm) of double-stranded nucleic acids that can address the problems in the prior art, a method for detecting mismatches in double-stranded nucleic acids or variations in sample nucleic acids relative to reference nucleic acids, and a kit for implementing these methods. An agent for increasing the melting temperature of double-stranded nucleic acids containing an amphiphilic copolymer comprising a specific cationic constituent unit (1) and a specific anionic constituent unit (2), a method for detecting nucleic acid mismatches and a method for detecting variations in sample nucleic acids relative to reference nucleic acids using the agent, and a nucleic acid mismatch detection kit and a variation detection kit containing the agent.

Description

[Technical field]

[0001] The present invention relates to an agent for increasing the melting temperature (Tm) of a double-stranded nucleic acid, a method for detecting mismatches in a double-stranded nucleic acid or variations in a sample nucleic acid relative to a reference nucleic acid using the agent, and a kit for implementing the agent. More specifically, the present invention relates to an agent for increasing the melting temperature (Tm) of a double-stranded nucleic acid containing an amphiphilic copolymer having a specific structure, a method for detecting mismatches in a double-stranded nucleic acid or variations in a sample nucleic acid relative to a reference nucleic acid using the agent, and a kit for implementing the agent. [Background technology]

[0002] Detection of mutations in genomic nucleic acids is widely performed to identify specific bacteria or viruses or to determine a person's susceptibility to disease or drug responsiveness. Recently, there is a demand to accurately, rapidly, and inexpensively detect single-base mutations in genomic nucleic acids.

[0003] As a method for detecting mutations in genomic nucleic acids, various methods have been developed, and for example, a method for detecting mismatches in sample nucleic acids relative to the reference nucleic acid is proposed by allowing a double-stranded nucleic acid composed of a reference nucleic acid and its complementary chain to coexist with a sample nucleic acid, and measuring the speed or rate at which the complementary chain is substituted for the sample nucleic acid (Patent Documents 1 to 3). In this method, by adding a cationic polymer such as polylysine or polyarginine as the main chain and a polymer grafted with dextran or polyethylene glycol as the side chain, the speed at which the complementary chain is substituted for the sample nucleic acid is promoted and the detection sensitivity is improved, and even a mismatch of one base can be detected. However, since this method detects the substitution of the complementary chain for the sample nucleic acid by the FRET method, it becomes necessary to label the reference nucleic acid and the complementary chain with a fluorescent dye, and it would be more convenient if this could be omitted.

[0004] As a method that can detect the variation of genomic nucleic acid without using nucleic acid labeled with fluorescent dye like FRET method, there is a method of detecting mismatch of double-stranded nucleic acid using melting temperature (Tm). Melting temperature (Tm) refers to the temperature at which 50% of double-stranded nucleic acid is denatured into single strands. Compared with fully matched double strands in a complementary relationship, the melting temperature (Tm) becomes lower in mismatched double strands that are not in a complementary relationship. Therefore, this phenomenon can be used to detect the variation of sample nucleic acid relative to the reference nucleic acid, or to detect the target sequence by the difference in nucleic acid amplification efficiency caused by this phenomenon. However, the change in melting temperature (Tm) caused by a mismatch of one base is considered to be about 1 to 3°C. In order to detect extremely slight variations in genomic nucleic acid using melting temperature (Tm), it becomes necessary to improve the sensitivity.

[0005] In this regard, a method that can analyze the melting curve and detect slight variations is performed (Patent Documents 4 and 5). However, in these methods, due to the need for strict temperature control, a high-precision thermostat becomes necessary, and a more practical method is desired. In addition, in the above-mentioned Patent Document 1, the Tm values ​​of a mismatched 20mer double-stranded nucleic acid with one base and a fully matched 20mer double-stranded nucleic acid are measured in the presence and absence of the above-mentioned cationic polymer, showing the Tm increase value caused by the presence of each cationic high molecular polymer. Unfortunately, the Tm increase value caused by the presence of a cationic polymer for the mismatched double-stranded nucleic acid and the fully matched double-stranded nucleic acid is about 15°C, and there is no significant difference between the two.

[0006] [Prior art literature]

[0007] [Patent Literature]

[0008] [Patent Document 1] International Publication No. 03 / 018841A1

[0009] [Patent Document 2] Japanese Patent Application No. 2001-78769

[0010] [Patent Document 3] Japanese Patent Application No. 2008-278779

[0011] [Patent Document 4] Japanese Patent Application No. 2005-58107

[0012] [Patent Document 5] Japanese Patent Application No. 2003-528626

[0013] [Patent Document 6] International Publication No. 2016 / 167320A1

[0014] [Patent Document 7] Patent No. 6242336

[0015] [Patent Document 8] Patent No. 5813263

[0016] [Patent Document 9] Patent No. 4731324

[0017] [Patent Document 10] Patent No. 4383178

[0018] [Patent Document 11] Patent No. 5030998

[0019] [Patent Document 12] Patent No. 4151751

[0020] [Patent Document 13] Japanese Patent Application Publication No. 2001-89496

[0021] [Patent Document 14] International Publication No. 2003 / 068795

[0022] [Patent Document 15] International Publication No. 2005 / 021570

[0023] [Patent Document 16] Patent No. 3756313

[0024] [Summary of the Invention]

[0025] [Problems to be solved by the invention]

[0026] The present invention aims to provide a novel agent for increasing the melting temperature (Tm) of double-stranded nucleic acids that can address the problems in the prior art as described above, a method for detecting mismatches in double-stranded nucleic acids or variations in sample nucleic acids relative to reference nucleic acids, and a kit for implementing these methods.

[0027]

Methods for solving the problem

[0028] As a result of intensive studies, the present inventors have discovered that when an amphiphilic copolymer containing a specific cationic constituent unit (1) and a specific anionic constituent unit (2) (hereinafter sometimes referred to as "specific amphiphilic copolymer") is added to a nucleic acid solution, the difference between the Tm increase value caused by the presence of the specific amphiphilic copolymer in a fully matched double-stranded nucleic acid in a complementary relationship and the Tm increase value caused by the presence of the specific amphiphilic copolymer in a mismatched double-stranded nucleic acid without a complementary relationship is amplified, thereby enabling mismatches to be detected even at a single base level, thereby completing the present invention.

[0029] That is, the present invention provides the following melting temperature increasing agent, nucleic acid mismatch detection method, sample nucleic acid variation detection method with respect to a reference nucleic acid, nucleic acid mismatch detection kit, and variation detection kit.

[0030] [1] An agent for increasing the melting temperature of a double-stranded nucleic acid, comprising an amphoteric copolymer, wherein the amphoteric copolymer comprises:

[0031] At least one cationic structural unit (1) selected from the following:

[0032] The structural unit (1-1) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0033]

Chemistry 1

[0034]

[0035] (Where R 1 represents a hydrogen atom, 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)

[0036] The structural unit (1-2) having a structure represented by the general formula (Ic) or the general formula (Id):

[0037]

Chemistry 2

[0038]

[0039] (Where R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms which may have a hydroxyl group, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, and X a- represents the counterion, and a represents the valence of the counterion)

[0040] The structural unit (1-3) having a structure represented by the general formula (Ie) or a structure of an acid addition salt thereof:

[0041]

Chemistry 3

[0042]

[0043] (Where R 4 and R 5 each independently represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an aralkyl group having 7 to 10 carbon atoms, or a cycloalkyl group having 5 to 6 carbon atoms which may have a hydroxyl group, and

[0044] A structural unit (1-4) having a structure represented by the general formula (If) or a structure of an acid addition salt thereof:

[0045]

Chemistry 4

[0046]

[0047] (R 6 represents a hydrogen atom or a methyl group, R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms, and n is an integer of 2 to 4), and

[0048] At least one anionic structural unit (2) selected from the following:

[0049] The structural unit (2-1) having a structure represented by the general formula (II-a):

[0050]

Chemistry 5

[0051]

[0052] (R 9 is hydrogen or methyl, and Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe)

[0053] The structural unit (2-2) having a structure represented by the general formula (II-b):

[0054]

Chemistry 6

[0055]

[0056] (wherein Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe)

[0057] The structural unit (2-3) having a structure represented by the general formula (II-c):

[0058]

Chemistry 7

[0059]

[0060] (wherein Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe), and,

[0061] The structural unit (2-4) having a structure represented by the general formula (II-d):

[0062]

Chemistry 8

[0063]

[0064] (R 10 is hydrogen or methyl, and Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe).

[0065] [2] The agent for increasing the melting temperature of a double-stranded nucleic acid according to [1], wherein

[0066] The cationic structural unit (1) is selected from the structural unit (1-1) and the structural unit (1-4),

[0067] The anionic structural unit (2) is selected from the structural unit (2-1) and the structural unit (2-4).

[0068] [3] The agent for increasing the melting temperature of a double-stranded nucleic acid according to [1] or [2], wherein the amphoteric copolymer further contains a nonionic structural unit (3).

[0069] [4] A method for detecting a nucleic acid mismatch between a sample nucleic acid and a probe nucleic acid, comprising:

[0070] (1) a step of measuring the melting temperature of the double-stranded nucleic acid of the probe nucleic acid and the nucleic acid having a base sequence complementary to the probe nucleic acid with or without adding the melting temperature-increasing agent for double-stranded nucleic acid according to any one of [1] to [3], and determining the increase in the melting temperature of the probe nucleic acid and the nucleic acid having a base sequence complementary to the probe nucleic acid due to the presence of the amphoteric copolymer (reference ΔTm);

[0071] (2) a step of measuring the melting temperature of the double-stranded nucleic acid of the sample nucleic acid and the probe nucleic acid with or without adding the melting temperature increasing agent described in any one of [1] to [3], and determining the increase in the melting temperature of the double-stranded nucleic acid of the sample nucleic acid and the probe nucleic acid caused by the presence of the amphoteric copolymer (sample ΔTm); and

[0072] (3) A step of determining the presence or absence of a nucleic acid mismatch based on a value (ΔΔTm) obtained by subtracting the sample ΔTm from the reference ΔTm.

[0073] [5] The method for detecting a nucleic acid mismatch according to [4], wherein the presence of a nucleic acid mismatch is determined when the ΔΔTm is 5.0°C or higher.

[0074] [6] A kit for detecting a nucleic acid mismatch between a sample nucleic acid and a probe nucleic acid, comprising:

[0075] The above-mentioned probe nucleic acid,

[0076] A nucleic acid having a base sequence complementary to the above-mentioned probe nucleic acid, and

[0077] The agent for increasing the melting temperature according to any one of [1] to [3].

[0078] [7] A method for detecting a variation between a sample nucleic acid and a reference nucleic acid, comprising:

[0079] (1) a step of measuring the melting temperature of a double-stranded nucleic acid of the reference nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid with or without adding the melting temperature-elevating agent described in any one of [1] to [3], and determining an increase in the melting temperature of the double-stranded nucleic acid of the reference nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid due to the presence of the amphoteric copolymer (reference ΔTm);

[0080] (2) a step of measuring the melting temperature of a double-stranded nucleic acid of the sample nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid with or without adding the melting temperature-elevating agent described in any one of [1] to [3], and measuring the increase in the melting temperature of the double-stranded nucleic acid of the sample nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid caused by the presence of the amphoteric copolymer (sample ΔTm); and

[0081] (3) A step of determining the presence or absence of a variation based on a value (ΔΔTm) obtained by subtracting the sample ΔTm from the reference ΔTm.

[0082] [8] The mutation detection method described in [7], wherein the presence of a mutation is determined when the ΔΔTm is 5.0°C or higher.

[0083] [9] A kit for detecting a variation between a sample nucleic acid and a reference nucleic acid, comprising:

[0084] The above-mentioned reference nucleic acid,

[0085] A nucleic acid having a base sequence complementary to the above-mentioned reference nucleic acid, and

[0086] The agent for increasing the melting temperature according to any one of [1] to [3].

[0087]

[10] Use of an amphiphilic copolymer in the manufacture of an agent for increasing the melting temperature of a double-stranded nucleic acid, the amphiphilic copolymer comprising:

[0088] At least one cationic structural unit (1) selected from the following:

[0089] The structural unit (1-1) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0090]

Chemistry 9

[0091]

[0092] (Where R 1 represents a hydrogen atom, 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)

[0093] The structural unit (1-2) having a structure represented by the general formula (Ic) or the general formula (Id):

[0094]

Chemistry 10

[0095]

[0096] (Where R 2 and R 3are each independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms which may have a hydroxyl group, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, and X a- represents the counterion, and a represents the valence of the counterion)

[0097] The structural unit (1-3) having a structure represented by the general formula (Ie) or a structure of an acid addition salt thereof:

[0098]

Chemistry 11

[0099]

[0100] (Where R 4 and R 5 each independently represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an aralkyl group having 7 to 10 carbon atoms, or a cycloalkyl group having 5 to 6 carbon atoms which may have a hydroxyl group, and

[0101] A structural unit (1-4) having a structure represented by the general formula (If) or a structure of an acid addition salt thereof:

[0102]

Chemistry 12

[0103]

[0104] (R 6 represents a hydrogen atom or a methyl group, R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms, and n is an integer of 2 to 4), and

[0105] At least one anionic structural unit (2) selected from the following:

[0106] The structural unit (2-1) having a structure represented by the general formula (II-a):

[0107]

Chemistry 13

[0108]

[0109] (R 9 is hydrogen or methyl, and Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe),

[0110] The structural unit (2-2) having a structure represented by the general formula (II-b):

[0111]

Chemistry 14

[0112]

[0113] (wherein Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe),

[0114] The structural unit (2-3) having a structure represented by the general formula (II-c):

[0115]

Chemistry 15

[0116]

[0117] (wherein Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe), and,

[0118] The structural unit (2-4) having a structure represented by the general formula (II-d):

[0119]

Chemistry 16

[0120]

[0121] (R 10 is hydrogen or methyl, and Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe).

[0122]

[11]

[10] The use described in claim 1 is characterized in that:

[0123] The cationic structural unit (1) is selected from the structural unit (1-1) and the structural unit (1-4),

[0124] The anionic structural unit (2) is selected from the structural unit (2-1) and the structural unit (2-4).

[0125]

[12] The use according to

[10] or

[11] , wherein the amphoteric copolymer further contains a nonionic structural unit (3).

[0126]

[13] A method for increasing the melting temperature of a double-stranded nucleic acid, comprising mixing an amphiphilic copolymer with the double-stranded nucleic acid in a solution, wherein the amphiphilic copolymer comprises:

[0127] At least one cationic structural unit (1) selected from the following:

[0128] The structural unit (1-1) having a structure represented by the general formula (Ia) or the general formula (Ib) or a structure of an acid addition salt thereof:

[0129]

Chemistry 17

[0130]

[0131] (Where R 1 represents a hydrogen atom, 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),

[0132] The structural unit (1-2) having a structure represented by the general formula (Ic) or the general formula (Id):

[0133]

Chemistry 18

[0134]

[0135] (Where R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms which may have a hydroxyl group, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, and X a- represents the counterion, a represents the valence of the counterion),

[0136] The structural unit (1-3) having a structure represented by the general formula (Ie) or a structure of an acid addition salt thereof:

[0137]

Chemistry 19

[0138]

[0139] (Where R 4 and R 5 each independently represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an aralkyl group having 7 to 10 carbon atoms, or a cycloalkyl group having 5 to 6 carbon atoms which may have a hydroxyl group, and

[0140] A structural unit (1-4) having a structure represented by the general formula (If) or a structure of an acid addition salt thereof:

[0141]

Chemistry 20

[0142]

[0143] (R 6 represents a hydrogen atom or a methyl group, R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms, and n is an integer of 2 to 4), and

[0144] At least one anionic structural unit (2) selected from the following:

[0145] The structural unit (2-1) having the structure represented by the general formula (II-a):

[0146]

Chemical formula 21

[0147]

[0148] (wherein R 9 is hydrogen or methyl, and Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe) for each carboxyl group bonded thereto),

[0149] The structural unit (2-2) having the structure represented by the general formula (II-b):

[0150]

Chemical formula 22

[0151]

[0152] (wherein Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe) for each carboxyl group bonded thereto),

[0153] The structural unit (2-3) having the structure represented by the general formula (II-c):

[0154]

Chemical formula 23

[0155]

[0156] (wherein Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe) for each carboxyl group bonded thereto), and,

[0157] The structural unit (2-4) having the structure represented by the general formula (II-d):

[0158]

Chemical formula 24

[0159]

[0160] (wherein R 10 is hydrogen or methyl, and Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe) for each carboxyl group bonded thereto).

[0161]

[14]

[13] The method according to the above, characterized in that,

[0162] The cationic structural unit (1) is selected from the structural unit (1-1) and the structural unit (1-4),

[0163] The anionic structural unit (2) is selected from the structural unit (2-1) and the structural unit (2-4).

[0164]

[15] The method according to

[13] or

[14] , wherein the amphoteric copolymer further contains a nonionic structural unit (3).

[0165] According to the present invention, the melting temperature (Tm) of double-stranded nucleic acid can be used to easily and highly sensitively detect double-stranded nucleic acid mismatches. Thus, the melting temperature (Tm) of double-stranded nucleic acid can be used to easily and highly sensitively detect the presence or absence of a variation in a sample nucleic acid relative to a reference nucleic acid (e.g., a wild-type genomic nucleic acid).

[0166] [Brief description of the attached drawings]

[0167]

Figure 1

[0168]

Figure 2

[0169]

Figure 3

[0170]

Figure 4

[0171]

Figure 5

[0172]

Figure 6

[0173]

Figure 7

[0174]

Figure 8

[0175]

Fig. 9

[0176]

Fig.10

[0177]

Fig.11

[0178]

Fig.12

[0179]

Fig.13

[0180]

Fig.14

[0181]

Fig.15

[0182]

Fig.16

[0183]

Fig.17

[0184]

Fig.18

[0185]

Fig.19

[0186]

Fig. 20

[0187]

Fig.21

[0188]

Fig. 22

[0189]

Fig.23

[0190]

Fig.24

[0191]

Fig.25

[0192]

Fig.26

[0193] The embodiments of the present invention are described in detail. However, the present invention is not to be understood as being limited to the following embodiments. The present invention relates to an agent for increasing the melting temperature (Tm) of a double-stranded nucleic acid containing an amphiphilic copolymer containing a specific constituent unit, a method for detecting mismatches in a double-stranded nucleic acid or a variation in a sample nucleic acid relative to a reference nucleic acid using the agent, and a kit for implementing these methods. These embodiments are described in detail below.

[0194] 【1. Agents that increase the melting temperature (Tm) of double-stranded nucleic acids】

[0195] One embodiment of the present invention relates to an agent for increasing the Tm of a double-stranded nucleic acid containing a specific amphiphilic copolymer. The agent for increasing the Tm of a double-stranded nucleic acid may consist only of a specific amphiphilic copolymer, or may contain other components as in an aqueous solution in which the specific amphiphilic copolymer is dissolved in a nuclease-free medium (e.g., water). The agent for increasing the Tm of a double-stranded nucleic acid increases the melting temperature of the double-stranded nucleic acid by more than 16.0°C, preferably more than 20.0°C. In particular, when the double-stranded nucleic acid is a non-matched double-stranded nucleic acid (fully matched double-stranded nucleic acid), the agent for increasing the Tm of a double-stranded nucleic acid increases the melting temperature of the fully matched double-stranded nucleic acid by more than 20.0°C, preferably more than 25.0°C, and particularly preferably more than 30.0°C.

[0196]

Specific amphoteric copolymer

[0197] The specific amphoteric copolymer contains a cationic structural unit (1) having a specific structure and an anionic structural unit (2) having a specific structure, and may be composed of only the cationic structural unit (1) and the anionic structural unit (2), or may optionally contain other structural units in addition to the cationic structural unit (1) and the anionic structural unit (2). Examples of the other structural units include the nonionic structural unit (3) described later, or a cationic or anionic structural unit having a structure that does not correspond to either the cationic structural unit (1) having a specific structure or the anionic structural unit (2) having a specific structure.

[0198] [Cationic structural unit (1)]

[0199] The cationic structural unit (1) constituting the specific amphoteric copolymer is at least one cationic structural unit selected from the following structural units (1-1), structural units (1-2), structural units (1-3) and structural units (1-4). Next, as shown in the general formula, the cationic structural unit (1) has an amino group as a cationic group in its structure, and the amino group is preferably a secondary amino group or a tertiary amino group from the viewpoint of achieving a good Tm value increase effect. The specific amphoteric copolymer may contain only one cationic structural unit (1) or two or more cationic structural units (1). When containing two or more cationic structural units (1), the two or more cationic structural units (1) may be a combination of structural units that are all classified as structural units (1-1), a combination of structural units that are all classified as structural units (1-2), a combination of structural units that are all classified as structural units (1-3), or a combination of structural units that are all classified as structural units (1-4), or a combination of structural units that are different from each other among the structural units (1-1) to (1-4). Since mismatches in double-stranded nucleic acids or variations in sample nucleic acids relative to reference nucleic acids can be more accurately detected in specific amphiphilic copolymers, the cationic structural unit (1) is particularly preferably selected from structural unit (1-1) and structural unit (1-4), and the cationic structural unit (1) is most preferably structural unit (1-1).

[0200]

Composition unit (1-1)

[0201] The structural unit (1-1) is a structural unit having a structure represented by the following general formula (Ia) or general formula (Ib) or a structure which is an acid addition salt thereof.

[0202]

Chemistry 25

[0203]

[0204] In the formula, R 1 R represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms which may have a hydroxyl group, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms. 1 It is preferably a hydrogen atom, a methyl group, an ethyl group or a benzyl group, and particularly preferably a hydrogen atom or a methyl group.

[0205] The constituent unit (1-1) is a structure of an inorganic acid salt or an organic acid salt, etc., which optionally has the structure represented by the above-mentioned structural formula (1-a) or (1-b), that is, a structure as an acid addition salt. When the specific amphoteric copolymer has the constituent unit (1-1), when the specific amphoteric copolymer is produced, it is preferred to use a diallylamine monomer having an addition salt from the viewpoint of production cost, etc. Since the process of removing the addition salt such as HCl from the polymer is cumbersome and also causes the increase in cost, the use of the constituent unit (1-1) of the addition salt type that can be produced without requiring such a process is also a preferred embodiment from the viewpoint of cost, etc. From the viewpoint of ease of acquisition or controllability of the reaction, the inorganic acid salt or organic acid salt in the constituent unit (1-1) of this embodiment is preferably a hydrochloride, a carboxylate, a sulfonate, or an alkyl sulfate ester salt, and is particularly preferably a hydrochloride.

[0206]

Constitutional Unit (1-2)

[0207] The structural unit (1-2) is a structural unit having a structure represented by the following general formula (Ic) or general formula (Id).

[0208]

Chemistry 26

[0209]

[0210] In the formula, R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms which may have a hydroxyl group, a cycloalkyl group having 5 to 10 carbon atoms, or an aralkyl group having 7 to 10 carbon atoms, and X a- R represents the counterion, and a represents the valence of the counterion. 2 and R 3 Each of R is independently preferably a hydrogen atom, a methyl group, an ethyl group or a benzyl group, and particularly preferably a methyl group or an ethyl group. 2 and R 3 At least one of is preferably a hydrogen atom.

[0211] In the counterion X a- There is no particular limitation in the invention. From the viewpoint of the ease of obtaining or the controllability of the reaction, chloride ion, carboxylate ion, sulfonate ion, or alkyl sulfate ion is preferred, and chloride ion or ethyl sulfate ion is particularly preferred. When manufacturing a specific amphoteric copolymer, from the viewpoint of manufacturing cost, a diallylamine monomer having a counter ion is preferably used. Since the process of removing the counter ion from the polymer is cumbersome and also causes the cost increase, the use of a specific amphoteric copolymer having a counter ion type constituent unit (1-2) that can be manufactured without requiring such a process is also a preferred embodiment from the viewpoint of cost, etc.

[0212]

Constituent units (1-3)

[0213] The constituent units (1-3) are constituent units having a structure represented by the following general formula (I-e) or a structure as its acid addition salt.

[0214]

Chemical formula 27

[0215]

[0216] In the formula, R 4 and R 5 each independently represent a hydrogen atom, an alkyl group having 1 to 12 carbon atoms optionally having a hydroxyl group, an aralkyl group having 7 to 10 carbon atoms, or a cycloalkyl group having 5 to 6 carbon atoms. As R 4 and R 5 The preferred alkyl group having 1 to 12 carbon atoms or aralkyl group having 7 to 10 carbon atoms can also be either linear or branched. As examples thereof, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, octyl, decyl, dodecyl, benzyl, etc. can be mentioned. In addition, as the preferred cycloalkyl group having 5 to 6 carbon atoms for R 4 and R 5 cyclopentyl and cyclohexyl can be mentioned, and it is not limited to these. R 4 and R 5 each independently are preferably a hydrogen atom, methyl, ethyl or benzyl, and particularly preferably a hydrogen atom or methyl. From the viewpoint of achieving a good effect of increasing the Tm value, etc., it is preferred that R 4 and R 5 do not both become hydrogen atoms at the same time.

[0217] There is no particular limitation on the type of addition salt when the constituent unit (1-3) is an acid addition salt of the structure represented by the general formula (I-e). From the viewpoints of availability or ease of controlling the reaction, etc., for example, hydrochloride, sulfate, phosphate, nitrate, sulfite, phosphite, nitrite, hydrobromide, acetate, amidosulfate, methanesulfonate, trifluoroacetate, p-toluenesulfonate, etc. can be used.

[0218] Among them, hydrochloride, sulfate, phosphate, and amidosulfate are preferred, and particularly preferred are hydrochloride, sulfate, phosphate, and amidosulfate of a structure derived from monoallylamine.

[0219]

Constituent units (1-4)

[0220] The constituent units (1-4) are constituent units having a structure represented by the following general formula (I-f) or a structure as its acid addition salt.

[0221]

Chemical formula 28

[0222]

[0223] Where R 6 represents a hydrogen atom or a methyl group, R 7 and R 8 Each independently represents an alkyl group having 1 to 4 carbon atoms, and n is an integer of 2 to 4. 6 It is preferably methyl, n is preferably 2 to 3, R 7 and R 8 Each is preferably a methyl group. When the constituent unit (1-4) is an acid addition salt of a structure represented by the general formula (If), there is no particular restriction on the type of the addition salt. From the viewpoint of availability or ease of reaction control, for example, hydrochloride, sulfate, phosphate, nitrate, sulfite, phosphite, nitrite, hydrobromide, acetate, amide sulfate, methanesulfonate, trifluoroacetate, p-toluenesulfonate, etc. can be used. Specifically, hydrochloride, sulfate, phosphate, and amide sulfate are preferred, and hydrochloride, sulfate, phosphate, and amide sulfate of a structure derived from monoallylamine are particularly preferred.

[0224] The ratio of the cationic structural unit (1) to the total structural units of the specific amphoteric copolymer is usually 10 to 70 mol%, preferably 15 to 60 mol%, and particularly preferably 20 to 55 mol%. When two or more cationic structural units (1) are contained, the above ratio is defined based on the total amount of the two or more cationic structural units (1).

[0225] [Anionic structural unit (2)]

[0226] The anionic structural unit (2) constituting the specific amphoteric copolymer used in the present invention is at least one anionic structural unit selected from the following structural unit (2-1), structural unit (2-2), structural unit (2-3) and structural unit (2-4). The specific amphoteric copolymer may contain only one anionic structural unit (2), or may contain two or more anionic structural units (2). When containing two or more anionic structural units (2), the two or more anionic structural units (2) may be a combination of structural units that are both classified as structural units (2-1), a combination of structural units that are both classified as structural units (2-2), a combination of structural units that are both classified as structural units (2-3), or a combination of structural units that are both classified as structural units (2-4), or may be a combination of structural units that are different from each other among the structural units (2-1) to (2-4). In particular, the anionic structural unit (2) is preferably selected from the structural unit (2-1) and the structural unit (2-4) because mismatches in double-stranded nucleic acids or variations in sample nucleic acids relative to reference nucleic acids can be more reliably detected in the specific amphiphilic copolymer.

[0227]

Constituent unit (2-1)

[0228] The constituent unit (2-1) is a constituent unit having a structure represented by the following general formula (II-a).

[0229]

Chemical formula 29

[0230]

[0231] In the formula, R 9 is hydrogen or methyl, and Y independently represents hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al, or 1 / 3Fe for each carboxyl group to which it is bonded. R 9 is preferably hydrogen, and Y is preferably hydrogen or Na. The constituent unit (2-1) is particularly preferably derived from maleic acid.

[0232]

Constituent unit (2-2)

[0233] The constituent unit (2-2) is a constituent unit having a structure represented by the following general formula (II-b).

[0234]

Chemical formula 30

[0235]

[0236] In the formula, Y independently represents hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al, or 1 / 3Fe for each carboxyl group to which it is bonded. Y is preferably hydrogen or Na.

[0237]

Constituent unit (2-3)

[0238] The constituent unit (2-3) is a constituent unit having a structure represented by the following general formula (II-c) or a structure as its acid addition salt.

[0239]

Chemical formula 31

[0240]

[0241] In the formula, Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe for each carboxyl group to which it is bonded. Y is preferably hydrogen or Na.

[0242]

Constituent unit (2-4)

[0243] The constituent unit (2-4) is a constituent unit having a structure represented by the following general formula (II-d).

[0244]

Chemistry 32

[0245]

[0246] In the formula, R 10 is hydrogen or methyl, and Y is independently hydrogen, Na, K, NH 4 , 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe. R 10 It is preferably hydrogen, and Y is preferably hydrogen or Na. The structural unit (2-4) is preferably derived from (meth)acrylic acid, and particularly preferably derived from acrylic acid.

[0247] As the anionic constituent unit (2) in the specific amphoteric copolymer, only one anionic constituent unit (2) may be used alone, or a plurality of anionic constituent units (2) having different structures may be used in combination. When a plurality of different anionic constituent units are used, each anionic constituent unit may have a different structure within the range represented by the same general structural formula (II-a), (II-b), (II-c) or (II-d), and optionally may have a different structure represented by different general structural formulas. In the former case, for example, represented by the general structural formula (II-a), a plurality of anionic constituent units (2) having different structures due to different elements of Y may be used. In the latter case, for example, an anionic constituent unit (2-1) having a structure represented by the structural formula (II-a) and another anionic constituent unit (2-4) having a structure represented by the structural formula (II-d) may be used.

[0248] Since the specific amphoteric copolymer contains the cationic constituent unit (1) and the anionic constituent unit (2), it is an amphoteric copolymer having both cationic and anionic properties. Although the mechanism by which the difference in the melting temperature of double-stranded nucleic acids from the case where there is no mismatch in the double-stranded nucleic acids is significantly increased by using the specific amphoteric copolymer containing the cationic constituent unit (1) having a specific structure and the anionic constituent unit (2) having a specific structure is unclear, if the cation density of the cationic constituent unit is too high, the binding between nucleic acids is over-strengthened, and the measurement of the melting temperature of the double-stranded nucleic acids becomes difficult, and if the cation density of the cationic constituent unit is too low, the increase in the melting temperature of the double-stranded nucleic acids is reduced, and the detection sensitivity of the mismatch is reduced, it is presumed that the positive charge of the cationic constituent unit (1) having a specific structure and the negative charge of the anionic constituent unit (2) having a specific structure moderately interfere with each other, and the binding between nucleic acids is moderately strengthened.

[0249] As described above, since mismatches in double-stranded nucleic acids or variations in reference nucleic acid pairs in sample nucleic acids can be detected more reliably, in the specific amphoteric copolymer, the cationic structural unit (1) is preferably selected from the structural unit (1-1) and the structural unit (1-4), and the anionic structural unit (2) may be selected from the structural unit (2-1) and the structural unit (2-4). It is particularly preferable to use a specific amphoteric copolymer having a structural unit (1) selected from the structural unit (1-1) and the structural unit (1-4), and having a structural unit (2-1) and the structural unit (2-4).

[0250] The total ratio of the cationic structural unit (1) and the anionic structural unit (2) in the total structural units of the specific amphoteric copolymer is not particularly limited, and is usually 25 mol% or more, preferably 30 to 90 mol%, more preferably 35 to 75 mol%, and particularly preferably 40 to 60 mol%. The ratio of the cationic structural unit (1) to the anionic structural unit (2) is also not particularly limited, and the molar ratio (cationic structural unit (1): anionic structural unit (2)) is usually 0.1:1 to 2:1, preferably 0.3:1 to 0.8:1, more preferably 0.4:1 to 1.2:1, and particularly preferably 0.5:1 to 1:1. The molar ratio of the cationic constituent unit (1) and the anionic constituent unit (2) in the specific amphoteric copolymer can be determined by reprecipitating the specific amphoteric copolymer with an organic solvent such as isopropyl alcohol or acetone, and analyzing the reprecipitate using a Perkin Elmer 2400II CHNS / O fully automatic elemental analyzer or an apparatus of equivalent performance in an appropriate mode corresponding to the structure of the above-mentioned constituent unit, when the structures of the cationic constituent unit (1) and the anionic constituent unit (2) are known. Furthermore, the molar ratio of the cationic constituent unit (1) and the anionic constituent unit (2) in the specific amphoteric copolymer can be determined by using helium gas as a carrier gas, measuring a solid sample in a tin capsule, dropping the sample 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. When the structures of the cationic constituent units (1) and the anionic constituent units (2) in the specific amphoteric copolymer are unknown, the structures of the respective constituent units are determined by a known method using 1H-NMR or 13C-NMR before the measurement using the above-mentioned elemental analysis device. Alternatively, the structure can be calculated from the amount of each monomer supplied during the production (copolymerization) of the specific amphoteric copolymer and the amount of each monomer remaining without collecting the specific amphoteric copolymer. Furthermore, since the ratio (molar ratio) of constituent units derived from each monomer in the specific amphoteric copolymer is roughly consistent with the feed composition (molar ratio) of each constituent unit, it is convenient to treat the mixing ratio of the monomers as the ratio (molar ratio) of the constituent units in this specification.

[0251] [Other constituent units]

[0252] The specific amphoteric copolymer may optionally have other constituent units in addition to the above-mentioned cationic constituent units (1) and anionic constituent units (2). Examples of other constituent units include nonionic constituent units (3) or cationic or anionic constituent units that do not correspond to any of the cationic constituent units (1) and anionic constituent units (2). Since the specific amphoteric copolymer preferably contains nonionic constituent units (3) because it is located between the cationic constituent units (1) and the anionic constituent units (2) and functions as a spacer between the two to adjust the interference between the positive load and the negative load of each constituent unit, it becomes possible to more accurately detect mismatches in double-stranded nucleic acids or variations in sample nucleic acids relative to reference nucleic acids. In addition, the nonionic constituent unit is preferably located between the cationic constituent units (1) and the anionic constituent units (2). In particular, when the cationic constituent unit is a structure represented by the above-mentioned general formula (If) or a structure as an acid addition salt thereof, since the nonionic constituent unit functions particularly effectively as a spacer, the specific amphoteric copolymer preferably contains nonionic constituent units (3).

[0253] [Nonionic structural unit (3)]

[0254] The nonionic constituent unit (3) in the present embodiment is a constituent unit derived from a nonionic monomer that can be copolymerized with the cationic constituent unit (1) and the anionic constituent unit (2), and there is no particular limitation other than this. Preferably, a constituent unit is used that is derived from a methacrylate monomer, an acrylate monomer, a methacrylamide monomer, an acrylamide monomer, sulfur dioxide, etc. More specifically, there can be mentioned 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, or sulfur dioxide. Particularly preferably, a constituent unit is used that is derived from acrylamide. The nonionic structural unit (3) can be introduced into the polymer by using a nonionic monomer as a monomer. When the specific amphoteric copolymer has the nonionic structural unit (3), the content of the nonionic structural unit (3) is not particularly limited. In addition, the appropriate amount varies depending on the type of the nonionic structural unit (3). The molar ratio of the nonionic structural unit (3) to the anionic structural unit (2) is preferably nonionic structural unit (3) / anionic structural unit (2) = 0.1 / 1 to 1 / 1, more preferably 0.2 / 1 to 0.8 / 1, more preferably 0.3 / 1 to 0.7 / 1, and particularly preferably 0.4 / 1 to 0.6 / 1. When the nonionic structural unit (3) is derived from a methacrylate monomer, an acrylate monomer, a methacrylamide monomer, or an acrylamide monomer, the above ratio is preferably 0.1 / 1 to 1 / 1, more preferably 0.2 / 1 to 0.8 / 1, more preferably 0.3 / 1 to 0.7 / 1, and particularly preferably 0.4 / 1 to 0.6 / 1. When the nonionic structural unit (3) is derived from sulfur dioxide, the above ratio is preferably 0.1 / 1 to 1 / 1, and particularly preferably 0.2 / 1 to 1 / 1.

[0255] [Method for producing specific amphoteric copolymer]

[0256] The method for producing the specific amphoteric copolymer is not particularly limited and the specific amphoteric copolymer can be produced by a method known in the prior art. For example, the specific amphoteric copolymer can be produced by copolymerizing a cationic monomer having a structure corresponding to the cationic constituent unit (1), an anionic monomer having a structure corresponding to the anionic constituent unit (2), and a nonionic monomer having a structure corresponding to the nonionic constituent unit (3) as desired, and other monomers.

[0257] The solvent used for copolymerizing the cationic monomer of the structure corresponding to the cationic structural unit (1) and the anionic monomer of the structure corresponding to the anionic structural unit (2) is not particularly limited, and may be an aqueous solvent or an organic solvent such as alcohol, ether, sulfoxide, amide, etc., preferably an aqueous solvent. The monomer concentration used for copolymerizing the cationic monomer of the structure corresponding to the cationic structural unit (1) and the anionic monomer of the structure corresponding to the anionic structural unit (2) varies depending on the type of monomer and the type of solvent used for copolymerization, and is generally 10 to 75% by mass in the case of an aqueous solvent. This copolymerization reaction is generally 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 and diazo.

[0258] 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.

[0259] [2. Method for detecting nucleic acid mismatch between sample nucleic acid and probe nucleic acid]

[0260] Another embodiment of the present invention relates to a method for detecting a nucleic acid mismatch between a sample nucleic acid and a probe nucleic acid, the method comprising:

[0261] (1) determining an increase in the melting temperature of a double-stranded nucleic acid of the probe nucleic acid and a nucleic acid having a base sequence complementary to the probe nucleic acid due to the presence of the specific amphiphilic copolymer (reference ΔTm);

[0262] (2) a step of determining an increase in the melting temperature of double-stranded nucleic acids of the sample nucleic acid and the probe nucleic acid due to the presence of the specific amphoteric copolymer (sample ΔTm); and

[0263] (3) A step of determining the presence or absence of a nucleic acid mismatch based on a value (ΔΔTm) obtained by subtracting the sample ΔTm from the reference ΔTm.

[0264] In step (1), the increase in melting temperature (reference ΔTm) is determined by measuring the nucleic acid melting temperature (Tm1) of the double-stranded nucleic acid of the probe nucleic acid and the nucleic acid having a base sequence complementary thereto in the presence of the specific amphiphilic copolymer and the melting temperature (Tm2) of the double-stranded nucleic acid of the probe nucleic acid and the nucleic acid having a base sequence complementary thereto in the absence of the specific amphiphilic copolymer, and calculating Tm1-Tm2. Similarly, in step (2), the increase in melting temperature (sample ΔTm) is determined by measuring the melting temperature (Tm1) of the double-stranded nucleic acid of the sample nucleic acid and the probe nucleic acid in the presence of the specific amphiphilic copolymer and the melting temperature (Tm2) of the double-stranded nucleic acid of the sample nucleic acid and the probe nucleic acid in the absence of the specific amphiphilic copolymer and calculating Tm1-Tm2.

[0265] In the present application, "nucleic acid" also includes any one of DNA and RNA. In addition, it also includes non-natural nucleic acids. Nucleic acids containing sequences complementary to sample nucleic acids and probe nucleic acids may include all nucleic acids such as genomic DNA, cDNA, genomic RNA, mRNA, rRNA, etc. In addition, nucleic acids containing sequences complementary to probe nucleic acids and probe nucleic acids may be natural nucleic acids or non-natural nucleic acids. Non-natural nucleic acids are DNA or RNA containing artificial nucleotides in a portion thereof. As artificial nucleotides, for example, BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), peptide nucleic acids (PHONA) with phosphate groups, or nucleic acids containing morpholino nucleic acids in at least a portion thereof, etc. (the details of these non-natural nucleic acids are, for example, recorded in patent documents 6 to 16, etc., and their contents are incorporated into the present application by reference).

[0266] The polymerization degree of the sample nucleic acid and the probe nucleic acid is not particularly limited, but is usually 4 to 100 mer, preferably 5 to 50 mer, more preferably 6 to 30 mer, further preferably 7 to 20 mer, and particularly preferably 8 to 15 mer from the viewpoint of detection sensitivity.

[0267] The GC content of the sample nucleic acid and the probe nucleic acid is not particularly limited, but is generally 30 to 80%, preferably 50 to 75%, from the viewpoint of detection sensitivity.

[0268] The melting temperature of double-stranded nucleic acid is usually determined using a nucleic acid aqueous solution, and the solvent is a nuclease-free aqueous medium, which is typically determined. In addition, the nucleic acid can be dissolved in a buffer, for example, a nucleic acid buffer used in a nucleic acid analysis reagent can be used. In addition, the nucleic acid aqueous solution can contain a surfactant in a range that does not affect the increase in the melting temperature (Tm) caused by the specific amphiphilic copolymer, and the surfactant used in the nucleic acid analysis reagent can be mentioned.

[0269] In the measurement of the melting temperature (Tm) of a double-stranded nucleic acid, the specific amphoteric copolymer may be contained in the solution at a concentration of usually 0.03 to 3.0% by mass, and preferably 0.2 to 2.0% by mass.

[0270] In one embodiment of the present invention, in addition to the specific amphoteric copolymer, MgCl 2 The known melting temperature (Tm) raising agent is contained in the reaction solution. 2 When the concentration is 5 mM or less, it is preferred that the concentration be 5 mM or less. In addition, a salt such as NaCl may be contained within a range that does not affect the increase in the melting temperature (Tm). In this case, the salt is preferably set to 300 mM or less.

[0271] In the measurement of the melting temperature (Tm) of double-stranded nucleic acid, the sample nucleic acid and the probe nucleic acid are usually measured at a concentration of 10 to 1000 μM, preferably 30 to 300 μM. The solution pH can be 3.0 to 11.0, preferably 5.0 to 9.0.

[0272] The temperature progression in the determination of the melting temperature (Tm) of double-stranded nucleic acid also varies depending on the method for detecting the denaturation of double-stranded nucleic acid. Basically, it is sufficient to increase the temperature slowly from the temperature at which the double strand is completely formed to cause the denaturation of the double-stranded nucleic acid. As a method for detecting the denaturation of double-stranded nucleic acid, there are methods that utilize the absorbance of the nucleic acid in the ultraviolet region (e.g., 260nm) accompanying the denaturation of the double-stranded nucleic acid, methods that utilize probe nucleic acids labeled with fluorescent dyes, enzymes, luminescent dyes, etc., or probe nucleic acids and nucleic acids having sequences complementary thereto, to detect the degree of formation of double-stranded nucleic acids (e.g., FRET method), and methods that utilize the reduction in fluorescence intensity accompanying the denaturation of double-stranded nucleic acids by collecting fluorescent dyes, etc. between nucleic acids when double-stranded nucleic acids are formed (e.g., intercalator-method). Although there is no need to prepare probe nucleic acids labeled with fluorescent dyes, etc., it is preferred that the fluorescent dyes, etc. be collected between nucleic acids when double-stranded nucleic acids are formed, and the reduction in fluorescence intensity accompanying the denaturation of double-stranded nucleic acids (e.g., intercalator-method) be utilized. Although it is not necessary to prepare probe nucleic acids labeled with fluorescent dyes, etc., it is preferred that the fluorescent dyes, etc. be collected between nucleic acids when double-stranded nucleic acids are formed, and the reduction in fluorescence intensity accompanying the denaturation of double-stranded nucleic acids (e.g., intercalator-method) be utilized. Furthermore, when referring to a specific numerical value of Tm in the present specification, unless otherwise specified, it refers to the numerical value measured by the method described in Example 1, and the specific numerical values ​​of reference ΔTm, sample ΔTm and ΔΔTm refer to the values ​​calculated from the Tm obtained in this way.

[0273] The sensitivity of detecting nucleic acid mismatches between the sample nucleic acid and the probe nucleic acid varies under conditions such as the length of the nucleic acid, the GC content of the nucleic acid, and the salt concentration. In the detection method of the present invention, when the degree of polymerization of the nucleic acid is 20 or less, even if it is a mismatch of one base, the reference ΔTm usually becomes 5.0°C or more higher than the sample ΔTm. Therefore, it is preferred to determine that a double-stranded nucleic acid mismatch exists when the reference ΔTm becomes 5.0°C or more higher than the sample ΔTm. From the viewpoint of reducing false positives, it is preferred, more preferred, to determine that a nucleic acid mismatch exists when the reference ΔTm is 7.5°C or more, and particularly preferred to determine that a nucleic acid mismatch exists when the reference ΔTm is 10.0°C or more.

[0274] [3. Method for detecting mutation of sample nucleic acid relative to reference nucleic acid]

[0275] As one embodiment of the above-mentioned method for detecting nucleic acid mismatches, the presence or absence of a variation in a sample nucleic acid relative to a reference nucleic acid (e.g., a wild-type genomic nucleic acid) can be detected. Specifically, as the probe nucleic acid in the above-mentioned detection method, the antisense strand of the wild-type genomic nucleic acid is used, and as the nucleic acid having a base sequence complementary to the probe nucleic acid, the sense strand of the wild-type genomic nucleic acid is used, and similarly to the above, by detecting the probe nucleic acid, i.e., the antisense strand of the wild-type genomic nucleic acid and the nucleic acid mismatch of the sample nucleic acid, the variation in the sample nucleic acid relative to the sense strand of the wild-type genomic nucleic acid can be detected. In other words, according to other embodiments of the present invention, a variation detection method for detecting a variation in a sample nucleic acid relative to a reference nucleic acid is provided, which comprises:

[0276] (1) a step of measuring an increase in the melting temperature (reference ΔTm) of the reference nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid caused by the presence of a specific amphiphilic copolymer,

[0277] (2) a step of measuring an increase in the melting temperature of the sample nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid caused by the presence of the specific amphiphilic copolymer (sample ΔTm); and

[0278] (3) A step of determining the presence or absence of a variation based on the difference (ΔΔTm) between the sample ΔTm and the reference ΔTm.

[0279] 【4. Nucleic acid mismatch detection kit and mutation detection kit】

[0280] In other embodiments of the present invention, a method for detecting a nucleic acid mismatch between the sample nucleic acid and the probe nucleic acid, and a kit for implementing the method for detecting a variation in a sample nucleic acid relative to a reference nucleic acid are provided. Specifically, in one embodiment, a kit for detecting a nucleic acid mismatch between a sample nucleic acid and a probe nucleic acid is provided, the kit comprising:

[0281] probe nucleic acid, and

[0282] A nucleic acid having a base sequence complementary to the above-mentioned probe nucleic acid, and

[0283] An agent for increasing the melting temperature (Tm) of a double-stranded nucleic acid containing a specific amphiphilic copolymer.

[0284] In another embodiment, there is provided a kit for detecting a variation in a sample nucleic acid relative to a reference nucleic acid, the kit comprising:

[0285] Reference nucleic acid,

[0286] A nucleic acid having a base sequence complementary to the above-mentioned reference nucleic acid, and

[0287] An agent for increasing the melting temperature (Tm) of a double-stranded nucleic acid containing a specific amphiphilic copolymer.

[0288] The details and preferred forms of the "probe nucleic acid", "nucleic acid having a base sequence complementary to the probe nucleic acid", "reference nucleic acid", "nucleic acid having a base sequence complementary to the reference nucleic acid" and "specific amphiphilic copolymer" in these kits are as described above. In addition to these, other components used in the determination of the melting temperature (Tm) of double-stranded nucleic acids, such as aqueous media, buffers, surfactants, salts, other melting temperature (Tm) elevating agents, reagents that can be used to detect double-stranded nucleic acids, etc., are also as described above. In particular, from the point of making nucleic acid mismatch detection and mutation detection easier, it is preferred that the reagent for detecting double-stranded nucleic acids contain an intercalator fluorescent dye.

[0289] [Example]

[0290] Hereinafter, the present invention will be described more specifically by way of Examples, but the present invention is not limited thereto.

[0291] [Example 1]

[0292] An aqueous polymer solution containing a dimethylaminopropylmethacrylamide hydrochloride-acrylamide-acrylic acid copolymer was used as a Tm-increasing agent for double-stranded nucleic acid, and the Tm increase amplitudes in mismatched double strands and fully matched double strands were compared to evaluate the copolymer for mismatch detection.

[0293] [1. Aqueous polymer solution (agent for increasing the Tm of double-stranded nucleic acid)]

[0294] As a Tm-increasing agent for double-stranded nucleic acid, a polymer aqueous solution containing 10.0% by mass of a dimethylaminopropylmethacrylamide hydrochloride-acrylamide-acrylic acid copolymer prepared by copolymerizing dimethylaminopropylmethacrylamide hydrochloride (monomer 1), acrylamide (monomer 2) and acrylic acid (monomer 3) under the following polymerization condition 1, wherein the molar ratio of constituent unit 1 derived from monomer 1, constituent unit 2 derived from monomer 2 and constituent unit 3 derived from monomer 3 is 1:1:2 and the pH is 7.0 was prepared.

[0295] Polymerization conditions 1: 16.15 g (equivalent to 0.04 mol) of dimethylaminopropylmethacrylamide hydrochloride (solid content concentration 51.2 mass %), 2.93 g (equivalent to 0.04 mol) of acrylamide (solid content concentration 97 mass %), 5.82 g (equivalent to 0.08 mol) of acrylic acid (solid content concentration 99 mass %), and 143.86 g of distilled water were added to a 300 mL four-necked flask equipped with a thermometer, a stirrer, and a cooling tube, and the temperature was raised to 60° C. A 28.5 mass % ammonium persulfate aqueous solution was added every 2 hours so that the amount of ammonium persulfate in the aqueous solution became 0.25, 0.5, 0.5, and 0.75 mol % respectively relative to the total amount of monomers, and the reaction was continued overnight.

[0296] [2. Reference nucleic acid, nucleic acid consisting of a complementary sequence, and nucleic acid having a mutation]

[0297] In order to evaluate whether a single-base variation in the nucleotide sequence of a reference nucleic acid can be detected, the following oligo DNA was used.

[0298]

Table 1

[0299]

[0300] In the base sequences of the oligoDNAs in the table, bases indicated in bold are mismatched positions.

[0301] 【3. Preparation of reaction solution】

[0302] The following reaction solutions 1 to 4 were prepared. The fluorescent dye in the fluorescent dye solution corresponds to the intercalator fluorescent dye.

[0303] [Table 2: Reaction solutions 1 to 4]

[0304]

[0305] The concentration of the polymer (agent for increasing the Tm of a double-stranded nucleic acid) in the reaction solutions 2 and 4 was 1.0% by mass.

[0306] 【4. Preparation of double-strand melting curve】

[0307] Each prepared reaction solution was placed in a StepOnePlus Real-Time PCR System (manufactured by Thermo Fisher Scientific), heated at 95°C for 15 seconds, and then kept at 10°C for 1 minute. Next, the temperature was raised to 95°C while fluorescence measurement was performed every 0.3 seconds to obtain a double-stranded melting curve, and then kept at 95°C for 15 seconds.

[0308] [Reference example]

[0309] For reference, a double-stranded melting curve was obtained by using a magnesium chloride solution, which is known as a Tm-increasing agent for double-stranded nucleic acids, instead of the above-mentioned polymer aqueous solution. Specifically, the following reaction solutions 5 and 6 were prepared instead of the above-mentioned reaction solutions 2 and 4, and double-stranded melting curves were obtained from each reaction solution in the same manner as in Example 1.

[0310] [Table 3: Reaction solutions 5 and 6]

[0311]

[0312] [Test results]

[0313] exist Figure 1 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Figure 2 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Figure 1 As shown in FIG. 1 , in the absence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 37.0°C. On the other hand, in the presence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 67.30°C, and an increase in the Tm value of 30.30°C was confirmed. Figure 2 As shown, in the absence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant strand and the complementary strand was 34.25°C. On the other hand, in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant strand and the complementary strand was 53.35°C, and an increase of 19.10°C in the Tm value was observed. As a result, a difference of 11.20°C was observed in the increase of the Tm value between the reference nucleic acid and the complementary strand and between the variant strand and the complementary strand in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid).

[0314] [Example 2]

[0315] In addition to using a diallylamine hydrochloride·maleic acid copolymer obtained by copolymerizing diallylamine hydrochloride (monomer 1) and maleic acid (monomer 2) using the following polymerization condition 2 as a Tm-increasing agent for double-stranded nucleic acid, in which the molar ratio of constituent unit 1 derived from monomer 1 and constituent unit 2 derived from monomer 2 is 1:1, a double-stranded melting curve is obtained from each reaction solution in the same manner as in Example 1, and the Tm increase between the reference nucleic acid and the complementary chain (reference ΔTm) and the Tm increase between the variant chain and the complementary chain (sample ΔTm) caused by the presence of the polymer (Tm-increasing agent for double-stranded nucleic acid) are determined therefrom, and the difference between the two (ΔΔTm) is calculated. Polymerization Condition 2: 160.07 g (0.80 mol) of 66.78% diallylamine hydrochloride, 78.45 g (0.80 mol) of anhydrous maleic acid, and 91.86 g of distilled water were added to a 500 mL four-mouth bottle equipped with a thermometer, a stirrer, and a cooling tube, and the internal temperature was raised to 50° C. A 28.5% by mass aqueous solution of ammonium persulfate was added only in such an amount that the amount of ammonium persulfate in the aqueous solution became 0.5% by mass relative to the total amount of monomers, and polymerization was started. The above-mentioned ammonium persulfate aqueous solution was added only in such an amount that the amount of ammonium persulfate became 0.5% by mass relative to the total amount of monomers after 4 hours, 1.0% by mass relative to the total amount of monomers after 20 and 26 hours, and 1.5% by mass relative to the total amount of monomers after 45 and 51 hours, respectively, and the reaction was allowed to proceed for 68 hours.

[0316] exist Figure 3 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Figure 4 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Figure 3 As shown in FIG. 1 , in the absence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 37.00°C. On the other hand, in the presence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 69.85°C, and an increase in the Tm value of 32.85°C was confirmed. Figure 4 As shown, in the absence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 34.25°C. On the other hand, in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 56.05°C, and an increase of 21.80°C in the Tm value was confirmed. As a result, a difference of 11.05°C in the increase of the Tm value in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid) was confirmed between the reference nucleic acid and the complementary chain and between the variant chain and the complementary chain.

[0317] [Example 3]

[0318] The double-stranded nucleic acid Tm-increasing agent was a diallylmethylamine-maleic acid copolymer prepared by copolymerizing diallylmethylamine (monomer 1) and maleic acid (monomer 2) under the following polymerization condition 3, wherein the molar ratio of the constituent unit 1 derived from the monomer 1 to the constituent unit 2 derived from the monomer 2 was 1:1. The double-stranded melting curve was obtained from each reaction solution in the same manner as in Example 1, and the Tm increase between the reference nucleic acid and the complementary strand (reference ΔTm) and the Tm increase between the variant strand and the complementary strand (sample ΔTm) caused by the presence of the polymer (double-stranded nucleic acid Tm-increasing agent) were determined, and the difference between the two (ΔΔTm) was calculated. Polymerization condition 3: 1.86 kg (19.0 mol) of anhydrous maleic acid and 0.34 kg of distilled water were charged into a 20 L four-mouth flask equipped with a thermometer, a stirrer, and a cooling tube, and 2.11 kg (19.0 mol) of diallylmethylamine (DAMA) was added dropwise under cooling. Thereafter, the internal temperature was raised to 50° C. A 28.5 mass % aqueous ammonium persulfate solution was added to initiate polymerization so that the amount of ammonium persulfate in the aqueous solution was 0.5 mass % relative to the total amount of monomers. 1.0 mass % was added after 3, 21, and 25 hours, and the reaction was continued overnight.

[0319] exist Figure 5 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Figure 6 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Figure 5 As shown in FIG. 1 , in the absence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 37.00°C. On the other hand, in the presence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 65.05°C, and an increase in the Tm value of 28.05°C was confirmed. Figure 6 As shown, in the absence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 34.25°C. On the other hand, in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 50.95°C, and an increase of 16.70°C in the Tm value was confirmed. As a result, a difference of 11.35°C in the increase of the Tm value in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid) was confirmed between the reference nucleic acid and the complementary chain and between the variant chain and the complementary chain.

[0320] [Example 4]

[0321] In addition to using a diallylamine hydrochloride-acrylamide-acrylic acid copolymer obtained by copolymerizing diallylamine hydrochloride (monomer 1), acrylamide (monomer 2) and acrylic acid (monomer 3) under the following polymerization condition 4 as a Tm-increasing agent for double-stranded nucleic acid, in which the molar ratio of constituent unit 1 derived from monomer 1, constituent unit 2 derived from monomer 2 and constituent unit 3 derived from monomer 3 is 1:1:2, an oligo-oligonucleotide double-strand melting test was performed. Polymerization condition 4: 20.43 g (equivalent to 0.1 mol) of diallylamine hydrochloride (solid content concentration 65.40 mass%) and 110.9 g of distilled water were added to a 300 mL four-mouth bottle equipped with a thermometer, a stirrer and a cooling tube, and the temperature was raised to 65°C. After adding a 28.5 mass % aqueous solution of ammonium persulfate so that the amount of ammonium persulfate in the aqueous solution becomes 2.0 mol % relative to the total amount of monomers, 30 minutes later, a solution of 7.11 g (equivalent to 0.1 mol) of acrylamide (solid content concentration 97 mass %), 14.56 g (equivalent to 0.2 mol) of acrylic acid (solid content concentration 99 mass %), and 21.15 g of distilled water was dropped over 3 hours, and the reaction was continued overnight.

[0322] exist Figure 7 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Figure 8 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Figure 7 As shown in FIG. 1 , in the absence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 38.40°C. On the other hand, in the presence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 67.68°C, and an increase in the Tm value of 29.28°C was confirmed. Figure 8 As shown, in the absence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 31.64°C. On the other hand, in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 53.39°C, and an increase of 21.75°C in the Tm value was confirmed. As a result, a difference of 7.53°C in the increase of the Tm value in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid) was confirmed between the reference nucleic acid and the complementary chain and between the variant chain and the complementary chain.

[0323] [Comparative Example 1]

[0324] In addition to using an allylamine polymer (product name: PAA-03, manufactured by NITTOBO MEDICAL) having a weight average molecular weight (Mw) of 3000 as determined by GPC as a Tm-increasing agent for double-stranded nucleic acids, double-stranded melting curves were obtained from each reaction solution in the same manner as in Example 1, from which the Tm increase between the reference nucleic acid and the complementary strand (reference ΔTm) and the Tm increase between the variant strand and the complementary strand (sample ΔTm) caused by the presence of the polymer (Tm-increasing agent for double-stranded nucleic acids) were determined, and the difference between the two (ΔΔTm) was calculated. Fig. 9 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Fig.10 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Fig. 9 and 10 As shown, in the presence of the above-mentioned polymer (agent for increasing the Tm of a double-stranded nucleic acid), no clear peak can be detected, and determination of the Tm value is difficult.

[0325] [Comparative Example 2]

[0326] In addition to using an allylamine polymer (product name: PAA-08, manufactured by NITTOBO MEDICAL) having a weight average molecular weight Mw of 8000 as determined by GPC as a Tm-increasing agent for double-stranded nucleic acids, double-stranded melting curves were obtained from each reaction solution in the same manner as in Example 1, from which the Tm increase between the reference nucleic acid and the complementary strand (reference ΔTm) and the Tm increase between the variant strand and the complementary strand (sample ΔTm) caused by the presence of the polymer (Tm-increasing agent for double-stranded nucleic acids) were determined, and the difference between the two (ΔΔTm) was calculated. Fig.11 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Fig.12 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Fig.11 and 12 As shown, in the presence of the above-mentioned polymer (agent for increasing the Tm of a double-stranded nucleic acid), no clear peak can be detected, and determination of the Tm value is difficult.

[0327] [Comparative Example 3]

[0328] In addition to using an allylamine polymer (product name: PAA-15C, manufactured by NITTOBO MEDICAL) having a weight average molecular weight Mw of 15,000 as determined by GPC as a Tm-increasing agent for double-stranded nucleic acids, double-stranded melting curves were obtained from each reaction solution in the same manner as in Example 1, from which the Tm increase between the reference nucleic acid and the complementary strand (reference ΔTm) and the Tm increase between the variant strand and the complementary strand (sample ΔTm) caused by the presence of the polymer (Tm-increasing agent for double-stranded nucleic acids) were determined, and the difference between the two (ΔΔTm) was calculated. Fig.13The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Fig.14 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Fig.13 and 14 As shown, in the presence of the above-mentioned polymer (agent for increasing the Tm of a double-stranded nucleic acid), no clear peak can be detected, and determination of the Tm value is difficult.

[0329] [Comparative Example 4]

[0330] In addition to using an allylamine hydrochloride-diallylamine hydrochloride copolymer as a Tm-increasing agent for double-stranded nucleic acid, which is obtained by copolymerizing allylamine hydrochloride (monomer 1) and diallylamine hydrochloride (monomer 2) under the following polymerization condition 5, wherein the molar ratio of the constituent unit 1 derived from monomer 1 and the constituent unit 2 derived from monomer 2 is 1:1, and the weight average molecular weight Mw obtained by GPC measurement is 100,000, an oligo-oligonucleotide double-strand melting test is performed. Polymerization condition 5: 245.24 g (1.50 mol) of 57.22 mass % allylamine hydrochloride and 307.31 g (1.50 mol) of 65.22 mass % diallylamine hydrochloride are added to a 1L four-mouth bottle equipped with a thermometer, a stirrer, and a cooling tube, and the temperature is raised to 60°C. A 30% by mass suspension of V-50 (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was added only in such a manner that the amount of V-50 in the suspension was 0.50% by mass relative to the total amount of monomers to start polymerization. 0.50% by mass was added after 3 hours, and 0.25% by mass was added after 24, 27, 48, 51, and 54 hours, and the reaction was continued overnight. Fig.15 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Fig.16 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Fig.15 and 16 As shown, in the presence of the above-mentioned polymer (agent for increasing the Tm of a double-stranded nucleic acid), no clear peak can be detected, and determination of the Tm value is difficult.

[0331] [Comparative Example 5]

[0332] In addition to using an allylamine hydrochloride-diallylamine hydrochloride copolymer obtained by copolymerizing allylamine hydrochloride (monomer 1) and diallylamine hydrochloride (monomer 2) under the following polymerization condition 6 as a Tm-increasing agent for double-stranded nucleic acid, wherein the molar ratio of the constituent unit 1 derived from monomer 1 to the constituent unit 2 derived from monomer 2 is 4:1, and the weight average molecular weight Mw measured by GPC is 20,000, an oligo-oligonucleotide double-strand melting test is performed. Polymerization condition 6: 490.48 g (3.00 mol) of 57.22 mass % allylamine hydrochloride and 153.66 g (0.75 mol) of 65.22 mass % diallylamine hydrochloride are added to a 1L four-mouth bottle equipped with a thermometer, a stirrer, and a cooling tube, and the temperature is raised to 60°C. A 30% by mass suspension of V-50 (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was added only in such a manner that the amount of V-50 in the suspension was 1.00% by mass relative to the total amount of monomers, and polymerization was started. 1.00% by mass was added after 24 hours, and 0.50% by mass was added after 48 hours, and the reaction was further allowed to proceed overnight. Fig.17 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Fig.18 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Fig.17 and 18 As shown, in the presence of the above-mentioned polymer (agent for increasing the Tm of a double-stranded nucleic acid), no clear peak can be detected, and determination of the Tm value is difficult.

[0333] [Comparative Example 6]

[0334] Double-stranded melting curves were obtained from each reaction solution in the same manner as in Example 1, except that 50 mol% acetylated polyallylamine having a weight average molecular weight Mw of 15,000 as determined by GPC under the following reaction condition 1 was used. The Tm increase between the reference nucleic acid and the complementary strand (reference ΔTm) and the Tm increase between the variant strand and the complementary strand (sample ΔTm) caused by the presence of the polymer (Tm-increasing agent for double-stranded nucleic acid) were determined, and the difference between the two (ΔΔTm) was calculated. Fig.19 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Fig. 20 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Fig.19 and 20As shown, in the presence of the above polymer (Tm-increasing agent for double-stranded nucleic acid), no clear peak can be detected, and it is difficult to determine the Tm value. Reaction Condition 1: 17.00 kg (45.11 mol) of 15.15 mass % allylamine polymer (product name: PAA-15C, manufactured by NITTOBOMEDICAL) was added to a 20L four-necked flask equipped with a thermometer, a stirrer, and a cooling tube. Thereafter, 2.37 kg (22.55 mol) of anhydrous acetic acid was added dropwise while cooling, and the mixture was reacted at room temperature overnight.

[0335] [Comparative Example 7]

[0336] In addition to using a polyacrylamide having a weight average molecular weight Mw of 20,000 as determined by GPC and polymerized under the following polymerization condition 7, a double-stranded melting curve was obtained from each reaction solution in the same manner as in Example 1, from which the Tm increase between the reference nucleic acid and the complementary chain (reference ΔTm) and the Tm increase between the variant chain and the complementary chain (sample ΔTm) caused by the presence of the polymer (Tm increase agent for double-stranded nucleic acid) were determined, and the difference (ΔΔTm) between the two was calculated. Polymerization condition 7: 186.59 g of distilled water was added to a 300 mL four-necked flask equipped with a thermometer, a stirrer, and a cooling tube, and the temperature was raised to 60°C. After adding 0.75 g of ammonium persulfate, a solution of 25.65 g (equivalent to 0.35 mol) of acrylamide (solid content concentration 97% by mass) and 36.55 g of distilled water was added dropwise over 5 hours, and the reaction was continued overnight. Fig.21 The double-stranded melting curves obtained from reaction solutions 1, 2, and 5 are shown. Fig. 22 The double-stranded melting curves obtained from reaction solutions 3, 4, and 6 are shown. Fig.21 As shown in FIG. 1 , in the absence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 38.4°C. On the other hand, in the presence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the complementary strand was 49.81°C, and an increase of 11.41°C in the Tm value was confirmed. Fig. 22 As shown in the figure, in the absence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 31.64°C. On the other hand, in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the complementary chain was 42.14°C, and an increase of 10.50°C in the Tm value was observed. As a result, a difference of only 0.91°C in the increase of the Tm value in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid) was observed between the reference nucleic acid and the complementary chain and between the variant chain and the complementary chain.

[0337] Next, the test results are comprehensively displayed.

[0338]

Table 4~6

[0339]

[0340]

[0341] [Example 5]

[0342] As a complementary strand to the reference nucleic acid, a partially BNA-constituted unnatural oligo DNA was used, and similar to Example 1, a dimethylaminopropyl methacrylamide hydrochloride·acrylamide·acrylic acid copolymer was evaluated for mismatch detection.

[0343] [1. Aqueous polymer solution (Tm increasing agent for double-stranded nucleic acid)]

[0344] As a Tm increasing agent for double-stranded nucleic acid, an aqueous polymer solution containing 10.0% by mass of the dimethylaminopropyl methacrylamide hydrochloride·acrylamide·acrylic acid copolymer used in Example 1 and having a pH of 7.0 was prepared.

[0345] [2. Reference nucleic acid, nucleic acid composed of complementary sequences, and nucleic acid having a mutation]

[0346] In order to evaluate the ability to detect a single-base mutation in the nucleotide sequence relative to the reference nucleic acid, the following oligo DNAs were used.

[0347] [Table 7]

[0348]

[0349]

[0350] In the base sequences of the respective DNA oligomers in the table, the bases shown in bold are the mismatch positions. In addition, the nucleotides containing the underlined bases are BNAs having the following structure.

[0351] [Chemical formula 33]

[0352]

[0353] [4. Preparation of double-stranded melting curve]

[0354] The following reaction solutions 1 to 4 were prepared.

[0355] [Table 8: Reaction solutions 1 to 4]

[0356]

[0357] The concentrations of the polymer (Tm increasing agent for double-stranded nucleic acid) in reaction solutions 2 and 4 were 1.0% by mass.

[0358] [4. Preparation of double-stranded melting curve]

[0359] Each prepared reaction solution was placed in a StepOnePlus Real-Time PCR System (manufactured by Thermo Fisher Scientific), heated at 95°C for 15 seconds, and then kept at 10°C for 1 minute. Next, the temperature was raised to 95°C while fluorescence measurement was performed every 0.3 seconds to obtain a double-stranded melting curve, and then kept at 95°C for 15 seconds.

[0360] [Test results]

[0361] exist Fig.23 The double-stranded melting curves obtained from reaction solutions 1 and 2 are shown. Fig.24 The double-stranded melting curves obtained from reaction solutions 3 and 4 are shown. Fig.23 As shown, in the absence of the above polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the BNA complementary chain was 63.74° C. On the other hand, in the presence of the above polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the BNA complementary chain was 92.95° C., and an increase in the Tm value of 29.21° C. was confirmed.

[0362] In addition, if Fig.24 As shown, in the absence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the BNA complementary chain was 62.40°C. On the other hand, in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the BNA complementary chain was 79.92°C, and an increase of 17.52°C in the Tm value was confirmed. As a result, a difference of 11.69°C was confirmed in the increase of the Tm value between the reference nucleic acid and the BNA complementary chain and between the variant chain and the BNA complementary chain in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid).

[0363] [Example 6]

[0364] In addition to using a polymer aqueous solution containing 10.0 mass % of the diallylamine hydrochloride·maleic acid copolymer used in Example 2 and having a pH of 7.0 as the Tm-increasing agent for double-stranded nucleic acid, a double-stranded melting curve was obtained from each reaction solution in the same manner as in Example 5, from which the Tm increase between the reference nucleic acid and the BNA complementary chain (reference ΔTm) and the Tm increase between the variant chain and the BNA complementary chain (sample ΔTm) caused by the presence of the polymer (Tm-increasing agent for double-stranded nucleic acid) were determined, and the difference (ΔΔTm) between the two was calculated.

[0365] exist Fig.25 The double-stranded melting curves obtained from reaction solutions 1 and 2 are shown. Fig.26 The double-stranded melting curves obtained from reaction solutions 3 and 4 are shown. Fig.25 As shown in FIG. 1 , in the absence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the BNA complementary strand was 63.74°C. On the other hand, in the presence of the above-mentioned polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the reference nucleic acid and the BNA complementary strand was 92.95°C, and an increase in the Tm value of 29.21°C was confirmed. Fig.26 As shown in the figure, in the absence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the BNA complementary chain was 62.40°C. On the other hand, in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid), the Tm value between the variant chain and the BNA complementary chain was 81.10°C, and an increase of 18.60°C in the Tm value was confirmed. As a result, a difference of 10.61°C was confirmed in the increase of the Tm value between the reference nucleic acid and the BNA complementary chain and between the variant chain and the BNA complementary chain in the presence of the polymer (agent for increasing Tm of double-stranded nucleic acid).

[0366] The test results of Examples 5 and 6 are summarized below.

[0367] [Table 9: Examples 5 and 6]

[0368]

Claims

1. An agent for increasing the melting temperature of a double-stranded nucleic acid, characterized in that Contains: (A) an amphoteric copolymer, wherein the amphoteric copolymer is composed of the following units: The cationic structural unit (1) is a structural unit (1-1) 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 a hydrogen atom, a methyl group or an ethyl group, The anionic structural unit (2) is a structural unit (2-1) having a structure represented by the general formula (II-a): In the formula, R 9 is hydrogen or methyl, Y is independently hydrogen, Na, K, NH4, 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe for each bonded carboxyl group, wherein the molar ratio of the cationic constituent unit (1): the anionic constituent unit (2) is 1:1; or (B) an amphoteric copolymer, wherein the amphoteric copolymer is composed of the following units: The cationic structural unit (1) is a structural unit (1-1) 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 a hydrogen atom, a methyl group or an ethyl group, The anionic structural unit (2) is a structural unit (2-4) having a structure represented by the general formula (II-d): In the formula, R 10 is hydrogen or methyl, Y is independently hydrogen, Na, K, NH4, 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe for each bonded carboxyl group, and A nonionic structural unit (3) derived from a methacrylamide monomer or an acrylamide monomer, The molar ratio of the cationic constituent unit (1): the anionic constituent unit (2) is 1:2, and wherein the molar ratio of the nonionic constituent unit (3) / anionic constituent unit (2) is 1 / 2; or (C) an amphoteric copolymer, wherein the amphoteric copolymer is composed of the following units: The cationic structural unit (1) is a structural unit (1-4) having a structure represented by the general formula (If) or a structure of an acid addition salt thereof: In the formula, R 6 is a hydrogen atom or a methyl group, R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms, n is an integer from 2 to 4, The anionic structural unit (2) is a structural unit (2-4) having a structure represented by the general formula (II-d): In the formula, R 10 is hydrogen or methyl, Y is independently hydrogen, Na, K, NH4, 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al or 1 / 3Fe for each bonded carboxyl group, and A nonionic structural unit (3) derived from a methacrylamide monomer or an acrylamide monomer, The molar ratio of the cationic constituent unit (1): the anionic constituent unit (2) is 1:2, and The molar ratio of the nonionic structural unit (3) / the anionic structural unit (2) is 1 / 2.

2. A method for detecting a nucleic acid mismatch between a sample nucleic acid and a probe nucleic acid, characterized in that: include: (1) a step of measuring the melting temperature of a double-stranded nucleic acid of the probe nucleic acid and a nucleic acid having a base sequence complementary to the probe nucleic acid with or without adding the melting temperature-increasing agent for double-stranded nucleic acid according to claim 1, and determining an increase in the melting temperature of the probe nucleic acid and the nucleic acid having a base sequence complementary to the probe nucleic acid due to the presence of the amphiphilic copolymer (reference ΔTm); (2) a step of measuring the melting temperature of the double-stranded nucleic acid of the sample nucleic acid and the probe nucleic acid with or without adding the melting temperature-elevating agent according to claim 1, and determining the increase in the melting temperature of the double-stranded nucleic acid of the sample nucleic acid and the probe nucleic acid caused by the presence of the amphoteric copolymer (sample ΔTm); and (3) A step of determining the presence or absence of a nucleic acid mismatch based on a value (ΔΔTm) obtained by subtracting the sample ΔTm from the reference ΔTm. 3 . The method for detecting a nucleic acid mismatch according to claim 2 , wherein when the ΔΔTm is 5.0° C. or higher, it is determined that a nucleic acid mismatch exists.

4. A nucleic acid mismatch detection kit between a sample nucleic acid and a probe nucleic acid, comprising: The probe nucleic acid, a nucleic acid having a base sequence complementary to the probe nucleic acid, and The melting temperature raising agent according to claim 1.

5. A variation detection method for detecting a variation of a sample nucleic acid relative to a reference nucleic acid, comprising: (1) a step of measuring the melting temperature of a double-stranded nucleic acid of the reference nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid with or without adding the melting temperature-elevating agent according to claim 1, and determining an increase in the melting temperature of the double-stranded nucleic acid of the reference nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid due to the presence of the amphiphilic copolymer (reference ΔTm); (2) a step of measuring the melting temperature of a double-stranded nucleic acid of the sample nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid with or without adding the melting temperature-elevating agent according to claim 1, and measuring an increase in the melting temperature of the double-stranded nucleic acid of the sample nucleic acid and a nucleic acid having a base sequence complementary to the reference nucleic acid caused by the presence of the amphiphilic copolymer (sample ΔTm); and (3) A step of determining the presence or absence of a variation based on a value (ΔΔTm) obtained by subtracting the sample ΔTm from the reference ΔTm. 6 . The mutation detection method according to claim 5 , wherein when the ΔΔTm is 5.0° C. or higher, it is determined that a mutation exists.

7. A kit for detecting a variation between a sample nucleic acid and a reference nucleic acid, comprising: The reference nucleic acid, a nucleic acid having a base sequence complementary to the reference nucleic acid, and The melting temperature raising agent according to claim 1.

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