Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions

Definitions

• the present invention relates to a nucleic acid adsorption inhibitor, a nucleic acid solution, and a nucleic acid amplification method.
• DNA is a type of nucleic acid, a biopolymer that serves as a carrier of genetic information in many organisms on Earth.
• Specific examples of such techniques for detecting, quantifying, and sequencing DNA include DNA detection techniques such as agarose gel electrophoresis, nucleic acid amplification techniques such as PCR and LAMP, nucleic acid quantification techniques that utilize these techniques, such as quantitative PCR and digital PCR, sequencing techniques such as the Sanger method and next-generation sequencing, and techniques that use these techniques on microarrays or biochips.
• non-specific adsorption of DNA often becomes a problem. That is, during the manipulation, storage, and transport of DNA, DNA is often adsorbed to the surfaces of containers, microchips, etc., particularly to the surfaces of plastic members, resulting in a decrease in the concentration of free DNA.
• target DNA is analyzed at extremely low concentrations, such as micromolar to attomole or less, and it is often difficult to obtain surplus DNA, so loss of DNA due to adsorption to the inner walls of containers, etc., is a serious problem.
• the same problem occurs with nucleic acids other than double-stranded DNA.
• Non-Patent Document 1 introduces a plastic tube product that has been specifically modified, and describes how using this product to prepare and store a nucleic acid concentration standard suppresses the adsorption of nucleic acids to the container, improves the nucleic acid yield, and improves the accuracy and sensitivity of applications that use the standard.
• Patent Documents 1 and 2 disclose a technique for suppressing nucleic acid adsorption by coating the surface of a container or biochip with a specific coating material, and a technique for suppressing nucleic acid adsorption by forming a layer containing a specific polymeric substance.
• Non-Patent Document 1 the approach of improving plastics as in Non-Patent Document 1 is inconvenient because it requires all components that will come into contact with nucleic acids to be replaced with components to which the technology has been applied in advance.
• the technologies described in Patent Documents 1 and 2 are inconvenient because it requires the technology to be applied in advance to all components that will come into contact with the nucleic acid.
• the present invention aims to provide a technology that achieves inhibition of nucleic acid adsorption by an approach that improves the nucleic acid solution.
• the objective is to provide a nucleic acid adsorption inhibitor that can be added to a nucleic acid solution to inhibit the nucleic acid from adsorbing to a container or component, a nucleic acid solution containing the same, and a nucleic acid amplification method that uses the nucleic acid solution.
• This approach also has the advantage of allowing a high degree of freedom in adjusting the concentration of the additive depending on the characteristics of the sample.
• a nucleic acid adsorption inhibitor comprising one or more polymers selected from the following Group A: [Group A] Polymer A1: a polymer consisting only of constitutional units derived from monomer a containing a phosphorylcholine group; Polymer A2: a copolymer containing a constitutional unit derived from the monomer a and a constitutional unit derived from a monomer b containing a carboxy group; and Polymer A3: a copolymer containing a constitutional unit derived from the monomer a and a constitutional unit derived from a monomer c containing an alkoxy-carbonyl group having 1 to 20 carbon atoms; However, Polymer A1 to Polymer A3 do not contain any structural unit containing a cationic functional group other than a phosphorylcholine group.
• nucleic acid adsorption inhibitor according to [1], wherein the monomer a is at least one selected from the group consisting of 2-methacryloyloxyethyl phosphorylcholine and 2-methacrylamidoethyl phosphorylcholine, and is preferably 2-methacryloyloxyethyl phosphorylcholine.
• monomer b is at least one selected from the group consisting of (meth)acrylic acid and crotonic acid, preferably (meth)acrylic acid, and more preferably methacrylic acid.
• nucleic acid adsorption inhibitor according to any one of [1] to [3], wherein the monomer c is an alkyl ester of methacrylic acid having 1 to 20 carbon atoms, preferably an alkyl ester of methacrylic acid having 1 to 6 carbon atoms, and more preferably butyl methacrylate.
• nucleic acid adsorption inhibitor according to any one of the above [1] to [4], wherein the weight average molecular weight of the polymer A1 is 10,000 to 2,000,000, preferably 100,000 to 2,000,000, and more preferably 500,000 to 1,500,000.
• 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.
• 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.
• nucleic acid adsorption inhibitor according to any one of [1] to [7], wherein the ratio of the structural units derived from the monomer a to the total structural units of the polymer A2 is 1 to 99 mol%, preferably 5 to 90 mol%, more preferably 10 to 60 mol%, and even more preferably 20 to 40 mol%.
• nucleic acid adsorption inhibitor according to any one of [1] to [9], wherein the polymer A2 is a copolymer consisting of only a structural unit derived from the monomer a and a structural unit derived from the monomer b.
• nucleic acid adsorption inhibitor according to any one of [1] to [10], wherein the ratio of the structural units derived from the monomer a to the total structural units of the polymer A3 is 10 to 90 mol %, preferably 20 to 70 mol %, and more preferably 25 to 50 mol %.
• nucleic acid adsorption inhibitor according to any one of [1] to [12], wherein the polymer A3 is a copolymer consisting of only a structural unit derived from the monomer a and a structural unit derived from the monomer c.
• nucleic acid adsorption inhibitor according to any one of [1] to [13], wherein the nucleic acid is a double-stranded nucleic acid, preferably a double-stranded DNA.
• nucleic acid solution comprising the nucleic acid adsorption inhibitor according to any one of [1] to [13] above, and nucleic acid.
• nucleic acid solution according to [15] above which is an aqueous solution of nucleic acid.
• nucleic acid solution according to [15] or [16] wherein the nucleic acid is a double-stranded nucleic acid, preferably a double-stranded DNA.
• a method for suppressing adsorption of nucleic acids in a nucleic acid solution to a member in contact with the nucleic acid solution comprising mixing the nucleic acid adsorption inhibitor according to any one of [1] to [13] above, the nucleic acid, and a solvent.
• the nucleic acid solution is an aqueous nucleic acid solution, and the solvent is water.
• the nucleic acid is a double-stranded nucleic acid, preferably a double-stranded DNA.
• the nucleic acid adsorption inhibitor of the present invention can be used to prevent free nucleic acids present in a solution to which it is added from being adsorbed to the surfaces of containers, microchips, etc. Therefore, for example, in various tests using nucleic acid samples as specimens, it is expected that the detection sensitivity will be improved and the storage stability of specimens and standards containing nucleic acids will be improved.
• nucleic acid adsorption inhibitor refers to an agent that is added to a nucleic acid solution in order to inhibit the adsorption of nucleic acid to the surface of a component that comes into contact with the solution in a container, microchip, or the like that handles the nucleic acid solution.
• the nucleic acid adsorption inhibitor of the present invention contains one or more polymers selected from the following Group A.
• Group A Polymer A1: a polymer consisting only of constitutional units derived from monomer a containing a phosphorylcholine group;
• Polymer A2 a copolymer containing a constitutional unit derived from the monomer a and a constitutional unit derived from a monomer b containing a carboxy group;
• Polymer A3 a copolymer containing a constitutional unit derived from the monomer a and a constitutional unit derived from a monomer c containing an alkoxy-carbonyl group having 1 to 20 carbon atoms;
• Polymer A1 to Polymer A3 do not contain any structural unit containing a cationic functional group other than a phosphorylcholine group.
• structural unit means a repeating unit in a polymer that is derived from a monomer. Therefore, “structural unit” does not include non-repeating structures in a polymer (for example, structures derived from a polymerization initiator, etc.).
• phosphorylcholine group refers to a monovalent group represented by the following formula (in the formula, * indicates the bond position):
• Examples of monomer a in the constituent units derived from monomer a contained in polymers A1 to A3 are not particularly limited as long as they contain a phosphorylcholine group and are polymerizable with monomers b and c.
• Preferred examples include compounds containing a phosphorylcholine group and a vinyl group, such as 2-(meth)acryloyloxyethyl phosphorylcholine, 2-(meth)acrylamidoethyl phosphorylcholine, alkyl phosphorylcholine allyl ether, and alkyl phosphorylcholine vinyl ether.
• the monomer a is more preferably 2-methacryloyloxyethyl phosphorylcholine or 2-methacrylamidoethyl phosphorylcholine, and further preferably 2-methacryloyloxyethyl phosphorylcholine.
• the monomer a may be used alone or in combination of two or more kinds.
• the monomer a may be a commercially available product.
• Polymer A1 is a polymer consisting only of structural units derived from monomer a.
• a polymer consisting only of structural units derived from monomer a means a polymer whose entire structural units consist only of structural units derived from monomer a
• structural units means repeating units in a polymer derived from a monomer. Therefore, “structural units” does not include non-repeating structures in a polymer (for example, structures derived from a polymerization initiator, etc.).
• Polymer A1 may be a homopolymer consisting only of structural units derived from one type of monomer a, or a copolymer consisting only of structural units derived from two or more types of monomers a.
• the weight average molecular weight of polymer A1 is not particularly limited, but from the viewpoint of the effect of suppressing nucleic acid adsorption, it is preferably from 10,000 to 2,000,000, more preferably from 100,000 to 2,000,000, and even more preferably from 500,000 to 1,500,000.
• the weight average molecular weight can be determined in terms of polyethylene glycol by gel filtration chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).
• Examples of monomer b in the structural unit derived from monomer b contained in polymer A2 are not particularly limited as long as it contains a carboxy group and is polymerizable with monomer a, and examples include compounds containing a carboxy group and a vinyl group, such as (meth)acrylic acid, crotonic acid, 3-methylcrotonic acid, angelic acid, tiglic acid, fumaric acid, maleic acid, itaconic acid, citraconic acid, etc. From the viewpoint of ease of polymerization reaction, monomer b is preferably (meth)acrylic acid or crotonic acid, more preferably (meth)acrylic acid, and even more preferably methacrylic acid. The monomer b may be used alone or in combination of two or more kinds. In addition, a commercially available product may be used as the monomer b.
• the ratio of the structural units derived from monomer b to all 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 still more preferably 60 to 80 mol%.
• the ratio of the constituent units derived from monomer a to the total constituent units of polymer A2 is preferably 1 to 99 mol%, more preferably 5 to 90 mol%, even more preferably 10 to 60 mol%, and even more preferably 20 to 40 mol%.
• polymer A2 is a copolymer consisting only of constituent units derived from monomer a and constituent units derived from monomer b.
• a copolymer consisting only of constituent units derived from monomer a and constituent units derived from monomer b means a copolymer whose total constituent units are composed only of constituent units derived from monomer a and constituent units derived from monomer b.
• the weight-average molecular weight of polymer A2 is not particularly limited, but from the viewpoint of the effect of inhibiting nucleic acid adsorption, it is preferably 50,000 to 2,000,000, more preferably 100,000 to 2,000,000, even more preferably 300,000 to 1,500,000, and even more preferably 300,000 to 1,000,000.
• Examples of monomer c in the structural unit derived from monomer c contained in polymer A3 are not particularly limited as long as it contains an alkoxy-carbonyl group having 1 to 20 carbon atoms and is polymerizable with monomer a.
• Examples include methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, undecyl (meth)acrylate, lauryl (meth)acrylate, tridecyl (meth)acrylate, myristyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, [0033]
• alkyl esters of methacrylic acid having 1 to 20 carbon atoms such as 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, myristyl methacrylate, pentadecyl methacrylate, cetyl methacrylate, margaryl methacrylate, stearyl methacrylate, nonadecyl methacrylate, and arachidyl methacrylate, are preferred; from the viewpoint of nucleic acid adsorption inhibitory effect, alkyl esters of methacrylic acid having 1 to 20 carbon atoms, such as methyl methacryl
• the proportion of the structural units derived from monomer c to all structural units of polymer A3 is preferably 10 to 90 mol %, more preferably 30 to 80 mol %, and even more preferably 50 to 75 mol %, from the viewpoint of the nucleic acid adsorption inhibitory effect.
• the ratio of the constituent units derived from monomer a to the total constituent units of polymer A3 is preferably 10 to 90 mol%, more preferably 20 to 70 mol%, and even more preferably 25 to 50 mol%, from the viewpoint of the nucleic acid adsorption inhibitory effect.
• Polymer A3 is particularly preferably a copolymer consisting only of constituent units derived from monomer a and constituent units derived from monomer c.
• a copolymer consisting only of constituent units derived from monomer a and constituent units derived from monomer c means a copolymer whose total constituent units are only composed of constituent units derived from monomer a and constituent units derived from monomer c.
• the weight-average molecular weight of polymer A3 is not particularly limited, but from the viewpoint of the effect 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.
• the polymers A2 and A3 may contain structural units derived from monomers other than the above-mentioned monomers (hereinafter referred to as "other monomers") within the scope of not impairing the effects of the present invention, provided that the other monomers contain only anionic, amphoteric, or neutral functional groups.
• the other monomers include glycerin mono(meth)acrylate, benzyl(meth)acrylate, and isobornyl(meth)acrylate.
• the proportion of the structural units derived from other monomers to the total structural units of the polymers A2 and A3 is preferably 40 mol % or less, more preferably 20 mol % or less, and even more preferably 10 mol % or less. It is even more preferable that the polymers A2 and A3 do not contain structural units derived from other monomers.
• the copolymer may be any of a random copolymer, an alternating copolymer, a block copolymer, a graft polymer, and a copolymer having two or more of these structures, but from the viewpoint of the manufacturability of the polymer, a random copolymer is preferred.
• the monomers used in preparing polymers A1 to A3 may be commercially available products or may be produced by known methods.
• Polymers A1 to A3 can be produced by known methods (e.g., the method described in WO 2018/216628, etc.).
• the nucleic acid adsorption inhibitor of the present invention can contain components other than the polymers A1 to A3 to the extent that the nucleic acid adsorption inhibitory effect of the polymers A1 to A3 is not impaired.
• the other components are not particularly limited, but may be appropriately selected from those exemplified as "other components" in the nucleic acid solution of the present invention described below.
• Polymers A1 to A3 can be easily used as nucleic acid adsorption inhibitors by being contained in a nucleic acid solution.
• Possible methods for incorporating the nucleic acid adsorption inhibitor of the present invention into a nucleic acid solution include 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 advance in a solvent such as a buffer solution that dissolves nucleic acids, and placing the nucleic acid adsorption inhibitor of the present invention in advance in a container in which the nucleic acid solution is prepared (for example, coating the inner surface of the container with the nucleic acid adsorption inhibitor of the present invention) and then adding the nucleic acid solution thereto to dissolve it.
• a solvent such as a buffer solution that dissolves nucleic acids
• the material of the container, member, etc. for which the nucleic acid adsorption inhibitor of the present invention is used to prevent adsorption of nucleic acid is preferably resin, more preferably polypropylene.
• the final concentration of the nucleic acid adsorption inhibitor of the present invention added to the nucleic acid solution is preferably 0.01 to 5 w/v%, more preferably 0.1 to 1 w/v%, and even more preferably 0.1 to 0.5 w/v%. If the amount added is too small, the effect of inhibiting nucleic acid adsorption may not be obtained, and if too much is added, problems such as reaction inhibition may occur when the nucleic acid solution is used in applications such as enzyme reactions.
• the type of nucleic acid to which the nucleic acid adsorption inhibitor of the present invention can be applied is not particularly limited, but is preferably a double-stranded nucleic acid (e.g., double-stranded DNA, double-stranded RNA, DNA:RNA hybrid), more preferably double-stranded DNA.
• the nucleic acid may be artificially synthesized by chemical synthesis, in vitro synthesis (e.g., reverse transcription reaction), PCR, or the like, or may be prepared by a known method from cells, microorganisms, viruses, etc.
• the cells, microorganisms, viruses, etc. may be collected from nature or the environment, or from humans, animals, or plants, or may be isolated and cultured.
• the present invention further provides a nucleic acid solution containing the nucleic acid adsorption inhibitor of the present invention and nucleic acid.
• the final concentration of the nucleic acid adsorption inhibitor in the nucleic acid solution of the present invention is 0.01 to 5 w/v %, more preferably 0.1 to 1 w/v %, and even more preferably 0.1 to 0.5 w/v %.
• the nucleic acid contained in the nucleic acid solution of the present invention is preferably a nucleic acid in which two or more molecular chains are associated, more preferably a double-stranded nucleic acid, and even more preferably double-stranded DNA.
• the nucleic acid may be artificially synthesized by, for example, chemical synthesis, in vitro synthesis (e.g., reverse transcription reaction), PCR, etc., or may be provided as a virus, bacterial body, cell, body fluid, tissue, etc., or a suspension thereof, or a nucleic acid extract prepared therefrom, etc.
• the virus, bacterial body, cell, body fluid, tissue, etc. may be collected from nature or the environment, or from humans, animals, or plants, or may be isolated and cultured.
• the concentration of the nucleic acid is appropriately determined depending on the application in which the nucleic acid is used.
• the nucleic acid solution of the present invention may contain other components to the extent that the effects of the present invention are not impaired.
• other components include polyols, polyethers, proteins, salts, buffer solutions, surfactants, solvents, biochemical reagents, dyes, preservatives, oils, and solid phase carriers.
• polyols examples include glycerol, sucrose, glucose, and the like.
• polyether is polyoxyethylene glycol.
• proteins include albumin, gelatin, casein, enzymes, and the like.
• Examples of the 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 the buffer solution include Tris-HCl buffer, Good's buffer, glycine buffer, borate buffer, TE buffer, TAE buffer, TBE buffer, and SSC buffer.
• Examples of the surfactant include polyoxyethylene alkyl ether, polyoxyethylene sorbitan monoalkyl ether, and alkyl betaine.
• the solvent may be water or an organic solvent.
• organic solvent include ethanol, propanol, isoamyl alcohol, glycerin, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, chloroform, and phenol.
• the solvent is preferably water, and the nucleic acid solution of the present invention is preferably an aqueous nucleic acid solution.
• the biochemical reagents include, for example, flavins.
• dyes include nucleic acid staining reagents such as ethidium bromide and SYBR TM Green I, fluorescent dyes such as ROX TM Dye, and colorants such as Orange G, bromophenol blue, and xylene cyanol FF.
• preservatives include sodium azide, paraoxybenzoic acid preparations, dehydroacetic acid preparations, and Proclin preparations.
• the oil may, for example, be mineral oil.
• the solid phase carrier include silica beads and magnetic beads.
• the nucleic acid solution of the present invention can be subjected to an enzyme reaction while still containing the nucleic acid adsorption inhibitor of the present invention.
• the enzymatic reaction include cleavage by a nuclease, reverse transcription by a reverse transcriptase, replication by a DNA polymerase, and nucleic acid amplification using these reactions.
• the enzymatic reaction in the present invention is preferably DNA or RNA replication, more preferably PCR.
• the present invention provides a method for suppressing the adsorption of nucleic acid in a nucleic acid solution to a member in contact with the nucleic acid solution, comprising mixing the nucleic acid adsorption inhibitor of the present invention, the nucleic acid, and a solvent.
• nucleic acid adsorption inhibitor of the present invention nucleic acid, and a solvent may be mixed
• a solution containing nucleic acid and a solvent may be mixed with the nucleic acid adsorption inhibitor of the present invention
• a solution containing the nucleic acid adsorption inhibitor of the present invention and a solvent may be mixed with the nucleic acid adsorption inhibitor.
• nucleic acid solution obtained by mixing the nucleic acid adsorption inhibitor of the present invention, nucleic acid, and a solvent is the same as the description of the nucleic acid solution of the present invention described above.
• Polymer A1-1, polymer A2-1, polymer A3-1 to polymer A3-3, which are within the scope of the present invention, and polymer Z-1, which is outside the scope of the present invention, were prepared as follows.
• the obtained polymers were dissolved in Water, Nuclease free (manufactured by Nippon Gene Co., Ltd., hereinafter referred to as "PW") to a concentration 10 times the final concentration described in each condition of the Examples and Comparative Examples described later, and the obtained polymer aqueous solutions were used.
• PW Nuclease free
• Polymer A1-1 which corresponds to polymer A1 was prepared in the following manner. 100 g of 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as "MPC") was weighed into a polymerization glass flask, 150 g of purified water was added to dissolve it, and 2.0 g of 4,4'-azobis(4-cyanovaleric acid) (hereinafter referred to as ACVA) was added to the obtained solution. This solution was heated to 70°C and stirred for 6 hours under a nitrogen atmosphere to carry out polymerization.
• MPC 2-methacryloyloxyethyl phosphorylcholine
• ACVA 4,4'-azobis(4-cyanovaleric acid
• the obtained polymerization solution was purified by dialysis using a semipermeable membrane with a molecular weight cutoff of 20,000, and then freeze-dried to obtain polymer A1-1.
• the weight average molecular weight of polymer A1-1 was 1,030,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• MAc methacrylic acid
• BMA butyl methacrylate
• AIBN 2,2'-azobis(isobutyronitrile
• the obtained polymerization solution was purified by dialysis using a semipermeable 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 90,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• SMA stearyl methacrylate
• MPC MPC
• BMA/GLM glycerin monomethacrylate
• the obtained polymerization solution was purified by dialysis using a semipermeable membrane with a molecular weight cutoff of 3,000, and then freeze-dried to obtain polymer A3-3.
• the weight average molecular weight of polymer A3-3 was 20,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• QA N,N,N-trimethyl-N-(2-hydroxy-3-methacryloyloxypropyl)-ammonium chloride
• the obtained polymerization solution was purified by dialysis using a semipermeable 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 40,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• ⁇ -HindIII digest (manufactured by Takara Bio Inc.) was used and added to a DNA solution described below so as to give a final concentration of 10 ng/ ⁇ L.
• 10X buffer 400 mM Tris-HCl buffer solution (containing 100 mM sodium chloride, 60 mM magnesium chloride, 10 mM calcium chloride, pH 7.9) was prepared.
• PP tubes self-supporting type
• the electrophoresis buffer and gel from 4) were used, the amount of sample applied was 5 ⁇ L, and electrophoresis was performed at 100 V for about 30 minutes.
• the gel was stained by immersing it in Midori Green Advance (manufactured by Nippon Genetics Co., Ltd.) diluted 10,000-fold with 1 ⁇ TAE for about 15 minutes, and then destained by immersing it in pure water for about 15 minutes.
• 9) Using FASdigi Compact (manufactured by Nippon Genetics Co., Ltd.), images were taken under BlueGreen LED irradiation, and the quantitative value of the 6.6 kbp band was calculated using ImageJ (National Institutes of Health, USA), and expressed as a relative value with the control set at 100. The results are summarized in Table 2.
• Examples 1-1 to 1-3 are examples in which polymers A1-1 to A3-1, respectively, were used as nucleic acid adsorption inhibitors. These results show that the use of the polymers of the present invention inhibits DNA adsorption to polypropylene tubes and microchips, and increases the amount of remaining DNA.
• Example 1-4 is an example in which polymer A3-2 is used as a nucleic acid adsorption inhibitor.
• Polymer A3-2 is an example of a polymer in which the alkyl group in monomer c, which contains an alkoxy-carbonyl group having 1 to 20 carbon atoms in polymer A3-1, is changed from butyl (carbon number 4) to stearyl (carbon number 18). It can be seen that even in this case, the DNA adsorption inhibitory effect is expressed.
• Example 1-5 is an example in which polymer A3-3 is used as a nucleic acid adsorption inhibitor.
• Polymer A3-3 is an example of a polymer in which polymer A3-1 further contains another monomer. It can be seen that even in this case, the DNA adsorption inhibitory effect is expressed.
• Example 2 An experiment similar to that in Experimental Example 1 was carried out using a Labolan screw vial No. 2 (manufactured by AS ONE, hereinafter referred to as "glass tube") instead of the PP tube. 1) Each DNA aqueous solution shown in Table 3 was prepared and dispensed into glass tubes. 2) Other conditions and procedures were the same as in Experimental Example 1. The results are shown in Table 3.
• Examples 2-1 to 2-3 and Comparative Example 2-1 are the results of experiments similar to those of Examples 1-1 to 1-3 and Comparative Example 1-1, respectively, conducted using glass tubes instead of PP tubes. These results show that by using the polymer of the present invention, it is possible to achieve inhibition of nucleic acid adsorption even when the container material is glass.
• the nucleic acid adsorption inhibitor of the present invention By simply adding the nucleic acid adsorption inhibitor of the present invention to a nucleic acid solution, the nucleic acid can be inhibited from being adsorbed to components in a container or the like that come into contact with the aqueous solution. Therefore, for example, in genetic testing in the fields of medicine, veterinary medicine, and forensic medicine, it is expected that loss due to adsorption to containers or the like during nucleic acid extraction can be reduced, i.e., recovery rates can be improved, and detection sensitivity can be improved. In addition, by inhibiting the decrease in effective concentration due to adsorption to containers or the like during storage and transportation of samples for genetic testing that contain nucleic acids, it is expected that storage stability and transportation stability can be improved.

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