Nucleic acid adsorption inhibitor, nucleic acid solution, and nucleic acid amplification method

Abstract

The present invention provides a nucleic acid adsorption inhibitor containing one or more polymers selected from the group A: Polymer A1: a polymer comprising only a structural unit derived from a phosphorylcholine group-containing monomer a; polymer A2: a copolymer containing a structural unit derived from the monomer a and a structural unit derived from a carboxyl group-containing monomer b. And polymer A3: a copolymer containing a structural unit derived from the monomer a and a structural unit derived from a monomer c containing an alkoxy-carbonyl group having 1 to 20 carbon atoms, where polymers A1 to A3 do not contain a structural unit containing a cationic functional group other than a phosphorylcholine group.

Description

Technical Field

[0001] This invention relates to nucleic acid adsorption inhibitors, nucleic acid solutions, and nucleic acid amplification methods. Background Technology

[0002] DNA (deoxyribonucleic acid) is a type of nucleic acid and a biological macromolecule that serves as the medium for genetic information in many organisms on Earth. As methods for detecting, quantifying, and determining the base sequence of such DNA, specific examples include DNA detection techniques such as agarose gel electrophoresis, nucleic acid amplification techniques such as PCR and LAMP, nucleic acid quantification techniques such as quantitative PCR and digital PCR using these techniques, base sequence determination techniques such as Sanger sequencing and next-generation sequencer, and techniques using these operations on microarrays and biochips.

[0003] At this point, in any of the above applications, non-specific adsorption of DNA often becomes a problem. That is, when handling, storing, or transferring DNA, DNA adsorbs onto the surfaces of containers, microchips, etc., especially onto the surfaces of plastic components, often leading to a decrease in the concentration of free DNA. In particular, when performing DNA detection, quantification, and base sequence analysis, not only are extremely low concentrations of target DNA (below micromolar to attomole) used as the analytical object, but it is also often difficult to obtain the remaining DNA. DNA loss due to adsorption on the inner wall of the container and other surfaces has also become a major problem. In addition, the same problems arise with nucleic acids other than double-stranded DNA.

[0004] Against this backdrop, various studies have been conducted on plastic modification techniques aimed at inhibiting the adsorption of nucleic acids on plastic containers. For example, Non-Patent Literature 1 describes a specially modified plastic test tube product, which demonstrates that when using this product to prepare and store nucleic acid concentration standards, nucleic acid adsorption on the container is inhibited, the yield of the finished nucleic acid product is increased, and the accuracy and sensitivity of the application of the standard can be improved.

[0005] In addition, patent documents 1 and 2 disclose techniques for inhibiting nucleic acid adsorption by coating containers and biochips with specific coating materials, and techniques for inhibiting nucleic acid adsorption by forming a layer containing specific polymeric substances. Existing technical documents Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2012-78365 Patent Document 2: Japanese Patent Application Publication No. 2006-258630 Non-patent literature

[0007] Non-patent literature 1: Eppendorf PCR Consumables-Compatibility Guide for PCR and qPCR Cyclers Summary of the Invention The problem the invention aims to solve

[0008] However, methods for improving plastics, such as those described in Non-Patent Document 1, require replacing all components that the nucleic acid is expected to come into contact with beforehand with components that are compatible with the technology, which is inconvenient. Similarly, the technologies described in Patent Documents 1 and 2 also require applying the technology to all components that the nucleic acid is expected to come into contact with beforehand, which is also inconvenient.

[0009] In view of the above-mentioned problems, the present invention aims to provide a technique for inhibiting nucleic acid adsorption by modifying a method on the nucleic acid solution side. Specifically, the aim is to provide a nucleic acid adsorption inhibitor that can inhibit the adsorption of nucleic acid on containers or materials by pre-adding it to the nucleic acid solution, a nucleic acid solution containing the inhibitor, and a nucleic acid amplification method using the nucleic acid solution. This method also has the advantages of allowing for adjustment of the added concentration according to the characteristics of the sample, providing a high degree of freedom. The means to solve the problem

[0010] [1] A nucleic acid adsorption inhibitor containing one or more polymers selected from group A below. [Group A] Polymer A1: A polymer consisting solely of structural units derived from monomers a containing phosphocholine groups; Polymer A2: a copolymer comprising structural units derived from monomer a and structural units derived from monomer b containing a carboxyl group; and Polymer A3: A copolymer comprising structural units derived from monomer a and structural units derived from monomer c containing alkoxy-carbonyl groups having 1 to 20 carbon atoms, wherein polymers A1 to A3 do not contain structural units containing cationic functional groups other than phosphocholine groups.

[0011] [2] According to the nucleic acid adsorption inhibitor described in [1], the monomer a is selected from at least one of the group consisting of 2-methacryloyloxyethyl phosphate choline and 2-methacrylamide ethyl phosphate choline, preferably 2-methacryloyloxyethyl phosphate choline. [3] According to the nucleic acid adsorption inhibitor described in [1] or [2], the monomer a is selected from at least one of the group consisting of (meth)acrylic acid and crotonic acid, preferably (meth)acrylic acid, more preferably methacrylic acid. [4] According to any one of [1] to [3], the nucleic acid adsorption inhibitor, wherein the monomer c is an alkyl ester of methacrylic acid with 1 to 20 carbon atoms, preferably an alkyl ester of methacrylic acid with 1 to 6 carbon atoms, and more preferably butyl methacrylate.

[0012] [5] The nucleic acid adsorption inhibitor according to any one of [1] to [4], wherein the weight-average molecular weight of polymer A1 is 10,000 to 2,000,000, preferably 100,000 to 2,000,000, more preferably 500,000 to 1,500,000. [6] According to any one of [1] to [5], the weight-average molecular weight of the polymer A2 is 50,000 to 2,000,000, preferably 100,000 to 2,000,000, more preferably 300,000 to 1,500,000, and even more preferably 300,000 to 1,000,000. [7] The nucleic acid adsorption inhibitor according to any one of [1] to [6], wherein the weight-average molecular weight of the polymer A3 is 5,000 to 200,000, preferably 10,000 to 200,000, more preferably 10,000 to 50,000, and even more preferably 30,000 to 50,000.

[0013] [8] The nucleic acid adsorption inhibitor according to any one of [1] to [7] has a ratio of structural units derived from monomer a to total structural units of polymer A2 of 1 to 99 mol%, preferably 5 to 90 mol%, more preferably 10 to 60 mol%, and even more preferably 20 to 40 mol%. [9] The nucleic acid adsorption inhibitor according to any one of [1] to [8] has a ratio of structural units derived from monomer b to total structural units of polymer A2 of 1 to 99 mol%, preferably 10 to 95 mol%, more preferably 40 to 90 mol%, and even more preferably 60 to 80 mol%.

[10] According to any one of [1] to [9], the nucleic acid adsorption inhibitor, the polymer A2 is a copolymer consisting only of structural units derived from monomer a and structural units derived from monomer b.

[0014]

[11] The nucleic acid adsorption inhibitor according to any one of [1] to

[10] has a ratio of structural units derived from monomer a to total structural units of polymer A3 of 10 to 90 mol%, preferably 20 to 70 mol%, more preferably 25 to 50 mol%.

[12] The nucleic acid adsorption inhibitor according to any one of [1] to

[11] has a ratio of structural units derived from the monomer c to the total structural units of the polymer A3 of 10 to 90 mol%, preferably 30 to 80 mol%, more preferably 50 to 75 mol%.

[13] The nucleic acid adsorption inhibitor according to any one of [1] to

[12] , wherein the polymer A3 is a copolymer consisting only of structural units derived from monomer a and structural units derived from monomer c.

[0015]

[14] The nucleic acid adsorption inhibitor according to any one of [1] to

[13] , wherein the nucleic acid is a double-stranded nucleic acid, preferably double-stranded DNA.

[0016]

[15] A nucleic acid solution containing the nucleic acid adsorption inhibitor and nucleic acid as described in any one of [1] to

[13] .

[16] The nucleic acid solution according to

[15] is an aqueous solution of nucleic acid.

[17] According to the nucleic acid solution described in

[15] or

[16] , the nucleic acid is a double-stranded nucleic acid, preferably double-stranded DNA.

[18] A nucleic acid amplification method using the nucleic acid solution described in any one of

[15] to

[17] .

[0017]

[19] A method for inhibiting the adsorption of nucleic acid in a nucleic acid solution on a material in contact with the nucleic acid solution, comprising the step of mixing the nucleic acid adsorption inhibitor described in any one of [1] to

[13] , the nucleic acid, and a solvent.

[20] According to the method described in

[17] , the nucleic acid solution is an aqueous nucleic acid solution, and the solvent is water.

[21] According to the method described in

[19] or

[20] , the nucleic acid is a double-stranded nucleic acid, preferably double-stranded DNA. The effects of the invention

[0018] When using the nucleic acid adsorption inhibitor of the present invention, it is possible to inhibit the adsorption of free nucleic acids present in a solution containing the nucleic acid adsorption inhibitor onto the surface of containers, microchips, etc. Therefore, for example, in various tests using nucleic acid samples as analytes, improvements in detection sensitivity, nucleic acid-containing analytes, and the preservation stability of standards can be expected. Detailed Implementation

[0019] The present invention will now be described in detail. Furthermore, the various descriptions in this specification may be combined with each other, except where explicitly stated otherwise.

[0020] In this specification, when a range of numerical values ​​is specified, the lower and upper limits of each range can be combined. For example, when it is specified that "preferably 10 to 100, more preferably 20 to 90", "preferably lower limit: 10" can be combined with "more preferably upper limit: 90" (that is, the numerical range of "10 to 90" is also within the scope of this specification).

[0021] [Nucleic acid adsorption inhibitor] In this invention, "nucleic acid adsorption inhibitor" refers to an agent added to the nucleic acid solution to inhibit the adsorption of nucleic acids on the surface of components in contact with the solution in containers, microchips, etc. that process nucleic acid solutions.

[0022] The nucleic acid adsorption inhibitor of the present invention contains one or more polymers selected from group A below. [Group A] Polymer A1: A polymer consisting solely of structural units derived from monomers a containing phosphocholine groups; Polymer A2: a copolymer comprising structural units derived from monomer a and structural units derived from monomer b containing a carboxyl group; and Polymer A3: A copolymer comprising structural units derived from monomer a and structural units derived from monomer c containing alkoxy-carbonyl groups having 1 to 20 carbon atoms. Among them, polymers A1 to A3 do not contain structural units with cationic functional groups other than phosphocholine groups.

[0023] In this specification, "structural unit" refers to a repeating unit in a polymer derived from a monomer. Therefore, "structural unit" does not include non-repeating structures in the polymer (e.g., structures derived from polymerization initiators, etc.).

[0024] In this specification, "phosphocholine" refers to the monovalent group shown in the following formula (in the following formula, * indicates the bonding position).

[0025] [Chemistry 1]

[0026] In this specification, "alkoxy-carbonyl group with 1 to 20 carbon atoms" refers to a group in which an alkoxy group with 1 to 20 carbon atoms is combined with a carbonyl group (-CO-), such as acetyl, propionyl, butyryl, valeryl, hexanoyl, heptayl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, hexadecanoyl, pentadecanoyl, octadecanoyl, nonadecanoyl, eicosanoyl, etc.

[0027] Examples of monomers a in the structural units derived from monomer a contained in polymers A1 to A3 are not particularly limited as long as they contain a phosphocholine group and can be polymerized with monomers b and c. For example, preferred examples include compounds containing a phosphocholine group and a vinyl group such as 2-(meth)acryloyloxyethyl phosphocholine, 2-(meth)acrylamidoethyl phosphocholine, alkyl phosphocholine allyl ether, and alkyl phosphocholine vinyl ether.

[0028] In this context, "(meth)acryloyloxy" essentially refers to acryloyloxy or methacryloyloxy. When multiple (meth)acryloyloxy groups may exist, "(meth)acryloyloxy" refers to acryloyloxy and / or methacryloyloxy. Similarly, "(meth)acrylamide," "(meth)acrylic acid," and "(meth)acrylate," described later, also have the same meaning as "(meth)acryloyloxy."

[0029] From the viewpoint of polymer storage stability and raw material accessibility, monomer a is more preferably 2-methacryloyloxyethyl phosphate choline or 2-methacrylamide ethyl phosphate choline, and even more preferably 2-methacryloyloxyethyl phosphate choline. Monomer 'a' can be used alone or in combination with two or more monomers. Furthermore, commercially available monomer 'a' can be used.

[0030] Polymer A1 is a polymer composed solely of structural units derived from monomer a. Furthermore, in this specification, "a polymer composed solely of structural units derived from monomer a" means a polymer whose total structural units consist solely of structural units derived from monomer a, and "structural unit" refers to a repeating unit in a polymer derived from a monomer. Therefore, "structural unit" does not include non-repeating structures in the polymer (e.g., structures derived from polymerization initiators). Polymer A1 can be a homopolymer composed solely of structural units derived from one type of monomer a, or a copolymer composed solely of structural units derived from two or more types of monomer a.

[0031] The weight-average molecular weight of polymer A1 is not particularly limited, but from the viewpoint of inhibiting nucleic acid adsorption, it is preferably 10,000 to 2,000,000, more preferably 100,000 to 2,000,000, and even more preferably 500,000 to 1,500,000. In addition, in this specification, the weight-average molecular weight can be determined, for example, by gel filtration chromatography using a system such as the EcoSEC system (manufactured by Tosoh Corporation) and converted to polyethylene glycol.

[0032] As an example of monomer b, a structural unit derived from monomer b contained in polymer A2, there are no particular limitations as long as it contains a carboxyl group and can polymerize with monomer a. Examples include compounds containing carboxyl and vinyl groups such as (meth)acrylic acid, crotonic acid, 3-methylcrotonic acid, angelic acid, citric acid, fumaric acid, maleic acid, itaconic acid, and citric acid. From the viewpoint of ease of polymerization, monomer b is preferably (meth)acrylic acid or crotonic acid, more preferably (meth)acrylic acid, and even more preferably methacrylic acid. Monomer b can be used alone or in combination with two or more monomers. In addition, commercially available monomer b can be used.

[0033] From the perspective of nucleic acid adsorption inhibition effect, the ratio of structural units derived from monomer b to the total structural units of polymer A2 is preferably 1 to 99 mol%, more preferably 10 to 95 mol%, even more preferably 40 to 90 mol%, and even more preferably 60 to 80 mol%. From the viewpoint of nucleic acid adsorption inhibition effect, the proportion of structural units derived from monomer a to the total structural units of polymer A2 is preferably 1 to 99 mol%, more preferably 5 to 90 mol%, even more preferably 10 to 60 mol%, and still more preferably 20 to 40 mol%. Polymer A2 is particularly preferably a copolymer composed only of structural units derived from monomer a and structural units derived from monomer b. Furthermore, in this specification, "a copolymer composed only of structural units derived from monomer a and structural units derived from monomer b" refers to a copolymer whose total structural units are composed only of structural units derived from monomer a and structural units derived from monomer b.

[0034] The weight-average molecular weight of polymer A2 is not particularly limited, but from the viewpoint of inhibiting nucleic acid adsorption, it is preferably 50,000 to 2,000,000, more preferably 100,000 to 2,000,000, further preferably 300,000 to 1,500,000, and even more preferably 300,000 to 1,000,000.

[0035] Examples of monomers c derived from monomer c in polymer A3 include any monomer c that contains an alkoxy-carbonyl group with 1 to 20 carbon atoms and can polymerize with monomer a. There are no particular limitations. Examples include: methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, heptyl methacrylate, octyl methacrylate, nonyl methacrylate, decyl methacrylate, undecyl methacrylate, lauryl methacrylate, tridecyl methacrylate, tetradecyl methacrylate, pentadecyl methacrylate, and so on. Alkyl esters of (meth)acrylic acid containing 1 to 20 carbon atoms, such as hexadecyl acrylate, stearyl acrylate, nonadecanyl acrylate, and eicosyl acrylate; alkyl esters of crotonic acid containing 1 to 20 carbon atoms; alkyl esters of 3-methylcrotonic acid containing 1 to 20 carbon atoms; alkyl esters of angelic acid containing 1 to 20 carbon atoms; alkyl esters of cisic acid containing 1 to 20 carbon atoms; alkyl esters of fumaric acid containing 1 to 20 carbon atoms; alkyl esters of maleic acid containing 1 to 20 carbon atoms; alkyl esters of itaconic acid containing 1 to 20 carbon atoms; and alkyl esters of citric acid containing 1 to 20 carbon atoms. From the viewpoint of polymer preservation stability, the preferred alkyl esters of methacrylic acid with 1 to 20 carbon atoms include methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, heptyl methacrylate, octyl methacrylate, nonyl methacrylate, decyl methacrylate, undecyl methacrylate, lauryl methacrylate, tridecyl methacrylate, tetradecyl methacrylate, pentadecyl methacrylate, hexadecyl methacrylate, heptyl methacrylate, stearyl methacrylate, nonadecanyl methacrylate, and eicosyl methacrylate. From the viewpoint of nucleic acid adsorption inhibition effect, the preferred alkyl esters of methacrylic acid with 1 to 6 carbon atoms include methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, and hexyl methacrylate. The most preferred alkyl ester is butyl methacrylate. Monomer C can be used alone or in combination with two or more monomers. Furthermore, commercially available monomer C can be used.

[0036] From the perspective of nucleic acid adsorption inhibition effect, the ratio of structural units derived from monomer c to the total structural units of polymer A3 is preferably 10-90 mol%, more preferably 30-80 mol%, and even more preferably 50-75 mol%. From the viewpoint of nucleic acid adsorption inhibition effect, the proportion of structural units derived from monomer a to the total structural units of polymer A3 is preferably 10-90 mol%, more preferably 20-70 mol%, and even more preferably 25-50 mol%. Polymer A3 is particularly preferably a copolymer composed only of structural units derived from monomer a and structural units derived from monomer c. Furthermore, in this specification, "a copolymer composed only of structural units derived from monomer a and structural units derived from monomer c" refers to a copolymer whose total structural units are composed only of structural units derived from monomer a and structural units derived from monomer c.

[0037] The weight-average molecular weight of polymer A3 is not particularly limited, but from the viewpoint of inhibiting nucleic acid adsorption, it is preferably 5,000 to 200,000, more preferably 10,000 to 200,000, even more preferably 10,000 to 50,000, and even more preferably 30,000 to 50,000.

[0038] Without impairing the effects of the present invention, polymers A2 and A3 may also contain structural units derived from monomers other than those described above (hereinafter referred to as "other monomers"). However, other monomers contain only anionic, amphoteric, or neutral functional groups. Other monomers include, for example, glycerol mono(meth)acrylate, benzyl (meth)acrylate, and isobornyl (meth)acrylate. The proportion of structural units derived from other monomers relative to the total structural units of polymers A2 and A3 is preferably 40 mol% or less, more preferably 20 mol% or less, and even more preferably 10 mol% or less. Polymers A2 and A3 are even more preferably free of structural units derived from other monomers.

[0039] When the polymer is a copolymer, the copolymer can be any of the following: random copolymer, alternating copolymer, block copolymer, graft polymer, or copolymer having two or more of these structures. From the viewpoint of polymer manufacturability, random copolymer is preferred.

[0040] The monomers used to formulate polymers A1 to A3 can be commercially available products or can be manufactured according to known methods. Polymers A1 to A3 can be manufactured according to known methods (e.g., the method described in International Publication No. 2018 / 216628).

[0041] The nucleic acid adsorption inhibitor of the present invention may contain components other than polymers A1 to A3, without impairing the nucleic acid adsorption inhibition effect. These other components are not particularly limited, and may be appropriately selected as components exemplified as "other components" in the nucleic acid solutions of the present invention described later.

[0042] Polymers A1 to A3 can be readily used as nucleic acid adsorption inhibitors by being contained in nucleic acid solutions.

[0043] As a method for making a nucleic acid solution contain the nucleic acid adsorption inhibitor of the present invention, there are methods such as adding polymers A1 to A3 to a prepared nucleic acid solution and dissolving them, dissolving the nucleic acid adsorption inhibitor of the present invention in a solvent such as a buffer solution to which the nucleic acid is to be dissolved beforehand, and loading the nucleic acid adsorption inhibitor of the present invention into a container to which the nucleic acid solution is to be prepared beforehand (for example, coating the inside of the container with the nucleic acid adsorption inhibitor of the present invention beforehand) and then loading the nucleic acid solution into it to dissolve it.

[0044] The material of the container, component, etc., used to prevent nucleic acid adsorption by the nucleic acid adsorption inhibitor of the present invention is preferably resin, and more preferably polypropylene.

[0045] The final concentration of the nucleic acid adsorption inhibitor of the present invention added to the nucleic acid solution is preferably 0.01–5 w / v%, more preferably 0.1–1 w / v%, and even more preferably 0.1–0.5 w / v%. If the amount added is too small, there is a possibility that the nucleic acid adsorption inhibition effect cannot be obtained; if the amount added is too large, there is a possibility that problems such as reaction inhibition may occur when the nucleic acid solution is used for applications such as enzyme reactions.

[0046] The type of nucleic acid for which the nucleic acid adsorption inhibitor of the present invention can be applied is not particularly limited, but double-stranded nucleic acids (e.g., double-stranded DNA, double-stranded RNA, DNA / RNA hybrid chains) are preferred, and double-stranded DNA is more preferred. The nucleic acid can be artificially synthesized through chemical synthesis, in vitro synthesis (e.g., reverse transcription), PCR, or other methods, or it can be formulated from cells, microorganisms, viruses, etc., using known methods. These cells, microorganisms, viruses, etc., can be substances extracted from nature or the environment, humans, or plants and animals, or they can be isolated / cultured substances.

[0047] [Nucleic Acid Solution] The present invention further provides a nucleic acid solution containing the nucleic acid adsorption inhibitor and nucleic acid of the present invention. The concentration of the nucleic acid adsorption inhibitor in the nucleic acid solution of the present invention, as described above, is ultimately 0.01–5 w / v, more preferably 0.1–1 w / v, and even more preferably 0.1–0.5 w / v.

[0048] The nucleic acid contained in the nucleic acid solution of the present invention is preferably a nucleic acid composed of two or more molecular chains associated together, more preferably a double-stranded nucleic acid, and even more preferably a double-stranded DNA.

[0049] The nucleic acid can be, for example, artificially synthesized nucleic acids through chemical synthesis, in vitro synthesis (e.g., reverse transcription), PCR, etc., or it can be a substance provided as a virus, bacterial cell, cell, body fluid, tissue, etc., or as a suspension of these or a nucleic acid extract prepared from these. In addition, the virus, bacterial cell, cell, body fluid, tissue, etc. can be substances extracted from nature or the environment, from humans or animals and plants, or they can be substances that have been isolated / cultured. The concentration of the nucleic acid can be appropriately determined based on the application using the nucleic acid.

[0050] Without impairing the effects of the present invention, the nucleic acid solution of the present invention may contain other components. Examples of other components include: polyols, polyethers, proteins, salts, buffer solutions, surfactants, solvents, biochemical reagents, pigments, preservatives, oils, and solid carriers.

[0051] Examples of salts include glycerol, sucrose, and glucose. Examples of polyethers include polyethylene glycol. Examples of proteins include albumin, gelatin, casein, and enzymes.

[0052] Examples of salts include: salts of amino acids, salts of peptides, alkali metal salts, alkaline earth metal salts, and salts of organic acids such as ethylenediaminetetraacetic acid. Examples of buffer solutions include: Tris hydrochloric acid buffer, Good's buffer, glycine buffer, borate buffer, TE buffer, TAE buffer, TBE buffer, SSC buffer, etc. Examples of surfactants include: polyoxyethylene alkyl ethers, polyoxyethylene sorbitol monoalkyl ethers, and alkyl betaine.

[0053] Examples of solvents include water and organic solvents. Examples of organic solvents include ethanol, propanol, isoamyl alcohol, glycerol, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, chloroform, and phenol. Water is preferred as the solvent, and the nucleic acid solution of this invention is preferably an aqueous nucleic acid solution.

[0054] As a biochemical reagent, flavins are an example. Examples of pigments include ethidium bromide and SYBR. TM Green I and other nucleic acid staining reagents, ROX TM Fluorescent dyes, orange-yellow G, bromophenol blue, xylene blue FF, and other colorants.

[0055] Examples of preservatives include sodium azide, p-hydroxybenzoic acid preparations, dehydroacetic acid preparations, and ProClin preparations. Examples of oils include mineral oil. Examples of solid-phase carriers include silicon beads and magnetic beads.

[0056] [Nucleic Acid Amplification Method] The nucleic acid solution of the present invention can be directly supplied to enzyme reactions while still containing the nucleic acid adsorption inhibitor of the present invention. Examples of enzymatic reactions include, for instance, nuclease-based cleavage, reverse transcription based on reverse transcriptase, DNA polymerase-based replication, and nucleic acid amplification using these methods. The enzymatic reaction referred to in this invention is preferably DNA or RNA replication, and more preferably PCR.

[0057] [Methods for inhibiting nucleic acid adsorption] This invention provides a method for inhibiting the adsorption of nucleic acids in a nucleic acid solution onto a component in contact with the nucleic acid solution, comprising the step of mixing the nucleic acid adsorption inhibitor of this invention, the nucleic acid, and a solvent. There are no particular limitations on the mixing; for example, (1) the nucleic acid adsorption inhibitor, nucleic acid, and solvent of this invention can be mixed; (2) a solution containing nucleic acid and a solvent and the nucleic acid adsorption inhibitor of this invention can be mixed; (3) a solution containing the nucleic acid adsorption inhibitor and a solvent of this invention and the nucleic acid adsorption inhibitor can be mixed. The description of the nucleic acid solution obtained by mixing the nucleic acid adsorption inhibitor, nucleic acid, and solvent of this invention (e.g., the type of nucleic acid to be used, the concentration of the nucleic acid adsorption inhibitor of this invention, and other components) is the same as the description of the nucleic acid solution of this invention described above. Example

[0058] The present invention will be specifically described below through examples, but the present invention is not limited thereto.

[0059] [Polymer Synthesis] Polymers A1-1, A2-1, A3-1 to A3-3 (within the scope of the present invention), and polymer Z-1 (outside the scope of the present invention) were prepared according to the following key points. The resulting polymers were dissolved in nuclease-free water (manufactured by NIPPON GENE Co., Ltd., hereinafter referred to as "PW") until a concentration of 10 times the final concentration described in each of the examples and comparative examples described later was reached, and the resulting aqueous solutions of polymers were used. The six polymers used in the formulation are summarized in Table 1.

[0060] [Synthesis example 1] Prepare polymer A1-1, which belongs to polymer A1, according to the following procedure. 100 g of 2-methacryloxyethyl phosphocholine (hereinafter referred to as "MPC") was weighed into a glass flask for polymerization and dissolved in 150 g of purified water. 2.0 g of 4,4'-azobis(4-cyanopentanoic acid) (hereinafter referred to as "ACVA") was added to the resulting solution. Polymerization was carried out by heating the solution to 70 °C and stirring under a nitrogen atmosphere for 6 hours. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane with a molecular weight cutoff of 20,000, followed by freeze-drying to obtain polymer A1-1. The weight-average molecular weight of polymer A1-1 was determined by GPC under the conditions described below and converted to polyethylene glycol as 1,030,000.

[0061] [Synthesis example 2] Prepare polymer A2-1, which belongs to polymer A2, according to the following procedure. 60 g of MPC and 40 g of methacrylic acid (hereinafter referred to as "MAc") (MPC / MAc = 30 / 70 molar ratio) were weighed into a glass flask for polymerization, and 567 g of purified water was added to dissolve them. 5.2 g of 4,4'-azobis(4-cyanopentanoic acid) (hereinafter referred to as "ACVA") was added to the resulting solution. Polymerization was carried out by heating the solution to 70°C and stirring under a nitrogen atmosphere for 6 hours. The resulting polymer solution was purified by dialysis using a semi-permeable membrane with a molecular weight cutoff of 20,000, and then freeze-dried to obtain polymer A2-1. The weight-average molecular weight of polymer A2-1 was determined by GPC under the conditions described below, and converted to polyethylene glycol as 370,000.

[0062] [Synthesis example 3] Prepare polymer A3-1, which belongs to polymer A3, according to the following procedure. 47 g of MPC and 53 g of butyl methacrylate (hereinafter referred to as "BMA") were weighed into a glass flask for polymerization (MPC / BMA = 30 / 70 molar ratio), and 900 g of ethanol (hereinafter referred to as "EtOH") was added to dissolve them. The mixture was heated to 60 °C. Polymerization was carried out by adding 2.4 g of 2,2'-azobis(isobutyronitrile) (hereinafter referred to as "AIBN") to the resulting solution under a nitrogen atmosphere and stirring for 7 hours. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane with a molecular weight cutoff of 3,000, and then freeze-dried to obtain polymer A3-1. The weight-average molecular weight of polymer A3-1 was determined by GPC under the conditions described below and converted to polyethylene glycol to 90,000.

[0063] [Synthesis example 4] Prepare polymer A3-2, which belongs to polymer A3, according to the following procedure. 78 g of MPC and 22 g of stearyl methacrylate (hereinafter referred to as "SMA") (MPC / SMA = 80 / 20) were weighed into a glass flask for polymerization. 667 g of EtOH was added to dissolve the mixture, and the solution was heated to 60°C. Polymerization was carried out by adding 3.3 g of AIBN to the resulting solution under a nitrogen atmosphere and stirring for 6 hours. The resulting polymer solution was purified by dialysis using a semi-permeable membrane with a molecular weight cutoff of 3,000, and then freeze-dried to obtain polymer A3-2. The weight-average molecular weight of polymer A3-2 was determined by GPC under the conditions described later, and converted to polyethylene glycol as 40,000.

[0064] [Synthesis example 5] Prepare polymer A3-3, which belongs to polymer A3, according to the following procedure. 57 g of MPC, 28 g of BMA, and 15 g of glycerol monomethacrylate (hereinafter referred to as "GLM") were weighed into a glass flask for polymerization (MPC / BMA / GLM = 40 / 40 / 20 (molar ratio)). 409 g of ethanol and 201 g of purified water were added to dissolve them. 1.9 g of AIBN was added to the resulting solution. Polymerization was carried out by heating the solution to 60°C and stirring under a nitrogen atmosphere for 5 hours. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane with a molecular weight cutoff of 3,000, followed by freeze-drying to obtain polymer A3-3. The weight-average molecular weight of polymer A3-3 was determined by GPC under the conditions described below, and converted to polyethylene glycol as 20,000.

[0065] [Synthesis example 6] Polymer Z-1, which is outside the scope of this invention, is prepared according to the following procedure. 74 g of MPC and 26 g of N,N,N-trimethyl-N-(2-hydroxy-3-methacryloyloxypropyl)ammonium chloride (hereinafter referred to as "QA") were weighed into a glass flask for polymerization (MPC / QA = 70 / 30 molar ratio), and 525 g of purified water was added to dissolve them. 0.9 g of 2,2'-azobis(2-methylpropionamide) dihydrochloride was added to the resulting solution. Polymerization was carried out by heating the solution to 70°C and stirring under a nitrogen atmosphere for 2 hours. The resulting polymerization solution was purified by dialysis using a semi-permeable membrane with a molecular weight cutoff of 20,000, and then freeze-dried to obtain polymer Z-1. The weight-average molecular weight of polymer Z-1 was determined by GPC under the conditions described below, and converted to polyethylene glycol as 40,000.

[0066] [GPC Measurement] The GPC determination of polymers A1-1, A2-1, A3-1 to A3-3 and Z-1 obtained in Synthetic Examples 1 to 6 was carried out under the following conditions. GPC System: EcoSEC System (manufactured by Tosoh Corporation) Chromatographic column: Shodex OHpak SB-802.5HQ (manufactured by Showa Denko Corporation) and SB-806MHQ (manufactured by Showa Denko Corporation) connected in series. Development solvent: 20 mM sodium phosphate buffer (pH 7.4) Detector: Differential refractive index detector Molecular weight standard: EasiVial PEG / PEO (manufactured by Agilent Technologies) Flow rate: 0.5 mL / min Column temperature: 40℃ Sample: Dilute the obtained polymer with the developing solvent to a final concentration of 0.1% by weight. Injection volume: 100μL

[0067] [Table 1]

[0068] [Experimental Example 1] A solution containing double-stranded DNA, prepared by adding polymers A1-1, A2-1, A3-1 to A3-3, or Z-1 obtained in Synthesis Examples 1-6, was placed in a propylene tube. After standing at room temperature, the solution was developed by agarose gel electrophoresis. The residual DNA in the solution was calculated by comparing the quantitative value of the double-stranded DNA with that of the control group. That is, the amount of DNA remaining on the inner surface of the propylene tube, the microchip using the solution, etc., that was not adsorbed was compared. 1) As DNA, λ-HindIII digest (manufactured by Takara Bio Co., Ltd.) was added to the DNA solution described below at a final concentration of 10 ng / μL. 2) Prepare a 400mM Tris-HCl buffer (containing 100mM sodium chloride, 60mM magnesium chloride, and 10mM calcium chloride, pH 7.9) as a 10X buffer. 3) Prepare the DNA aqueous solutions shown in Table 2 and inject them into 2 mL of polypropylene screw cap tubes (self-standing type) (manufactured by AS ONE, hereinafter referred to as "PP tubes"). 4) Let it stand at room temperature overnight. 5) As an electrophoresis buffer, prepare a buffer (1X TAE) by diluting 50X TAE (manufactured by NIPPON GENE Co., Ltd.) according to the instructions. As an electrophoresis gel, add 1X TAE at a ratio of 100mL to 1g of agarose S (manufactured by NIPPON GENE Co., Ltd.), boil it to dissolve, and prepare a solidified gel (1% gel). 6) Add 1 / 6 of the 6X loading buffer and Triple Dye (manufactured by NIPPON GENE Co., Ltd.) to each of the internal solutions after 4) and mix them to obtain the electrophoresis sample. 7) Develop the sample from 6) using agarose gel electrophoresis. Additionally, use the electrophoresis buffer and gel from 4), applying 5 μL of sample, and electrophoresis at 100V for approximately 30 minutes. 8) The staining of the gel was performed by immersing it in Midori Green Advance (manufactured by Nippon Genetics Co., Ltd.) diluted 10,000 times with 1X TAE for about 15 minutes, followed by immersion in pure water for about 15 minutes to remove the stain. 9) Images were taken using the FASdigi Compact (manufactured by Nippon Genetics Co., Ltd.) under BlueGreen LED illumination. For 6.6 kbp bands, quantification was calculated using ImageJ (National Institutes of Health). Results are expressed as relative values ​​with a control of 100. Results are summarized in Table 2.

[0069] [Table 2]

[0070] Examples 1-1 to 1-3 illustrate the use of polymers A1-1 to A3-1 as nucleic acid adsorption inhibitors. The results show that by using the polymers of the present invention, DNA adsorption on polypropylene tubes and microchips is inhibited, and the amount of residual DNA increases.

[0071] Examples 1-4 illustrate the use of polymer A3-2 as a nucleic acid adsorption inhibitor. Polymer A3-2 is an example of a polymer in which the alkyl group (c) of the monomer containing an alkoxy-carbonyl group with 1 to 20 carbon atoms in polymer A3-1 is changed from butyl (4 carbon atoms) to stearyl (18 carbon atoms). It is known that DNA adsorption inhibition is also observed in this case.

[0072] Examples 1-5 illustrate the use of polymer A3-3 as a nucleic acid adsorption inhibitor. Polymer A3-3 is an example of polymer A3-1 further containing other monomers. It is known that DNA adsorption inhibition is also observed in this case.

[0073] [Experimental Example 2] The same experiment as in Experiment 1 was conducted using LABORAN threaded tube bottle No. 2 (manufactured by AS ONE, hereinafter referred to as "glass tube") instead of PP tube. 1) Prepare the aqueous solutions of each DNA shown in Table 3 and inject them into glass tubes respectively. 2) The other conditions and order were the same as in Experiment 1. The results are shown in Table 3.

[0074] [Table 3] ※3 Any copolymer was added to a final concentration of 0.1% (w / v). ※4 is expressed as a relative value with a reference value of 100.

[0075] Examples 2-1 to 2-3 and Comparative Example 2-1 are the results of the same experiments performed as in Examples 1-1 to 1-3 and Comparative Example 1-1, respectively, using glass tubes instead of PP tubes. These results demonstrate that, when using the polymer of the present invention, nucleic acid adsorption inhibition can be achieved even when the container material is glass. Industrial availability

[0076] By simply adding the nucleic acid adsorption inhibitor of the present invention to the nucleic acid solution beforehand, the adsorption of the nucleic acid on components in contact with the aqueous solution within containers or the like can be suppressed. Therefore, in gene testing in fields such as medicine, veterinary medicine, and forensic medicine, it is expected to reduce losses due to adsorption on containers or the like during nucleic acid extraction, i.e., improve recovery rates and detection sensitivity. Furthermore, when storing and transporting analytes for gene testing containing nucleic acids, by suppressing the decrease in effective concentration caused by adsorption on containers or the like, it is expected to improve storage stability and transport stability.

[0077] This application is based on Japanese Patent Application No. 2023-055743, the entire contents of which are contained in this specification.

Claims

1. A nucleic acid adsorption inhibitor comprising one or more polymers selected from group A below, Group A: Polymer A1: A polymer consisting solely of structural units derived from monomers a containing phosphocholine groups; Polymer A2: a copolymer comprising structural units derived from monomer a and structural units derived from monomer b containing a carboxyl group; and Polymer A3: A copolymer comprising structural units derived from monomer a and structural units derived from monomer c containing alkoxy-carbonyl groups having 1 to 20 carbon atoms. in, Polymers A1 to A3 do not contain structural units with cationic functional groups other than phosphocholine groups.

2. A nucleic acid solution comprising the nucleic acid adsorption inhibitor of claim 1 and nucleic acid.

3. A nucleic acid amplification method using the nucleic acid solution described in claim 2.

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