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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F130/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
  • C08F130/02—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
  • C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus

Definitions

• the present invention relates to a dry composition for amplifying nucleic acids and a method for amplifying nucleic acids using the same.
• Nucleic acid amplification is known as a reaction in which a nucleic acid is used as a template to newly synthesize a nucleic acid complementary to the nucleic acid.
• a nucleic acid amplification reaction in addition to the nucleic acid that serves as the template, multiple nucleic acid amplification reagents are required, such as oligonucleotides called primers and enzymes. It is known that these nucleic acid amplification reagents deteriorate significantly at room temperature (e.g., 15°C to 25°C) or under refrigerated conditions due to a decrease in enzyme activity, etc. Drying the nucleic acid amplification reagent is known as a method for preventing this deterioration.
• Patent Documents 1 to 3 The most commonly used drying method is freeze-drying (see, for example, Patent Documents 1 to 3).
• Other drying methods such as Patent Document 4, describe a method of drying at room temperature and atmospheric pressure (air drying method) and a method of drying at a pressure above room temperature and up to 90% of atmospheric pressure (evaporation drying method).
• the present invention has been made in consideration of the above problems, and aims to provide a dry composition for amplifying nucleic acids that has excellent storage stability.
• a dry composition for amplifying nucleic acid comprising a polymer containing a constitutional unit derived from a monomer represented by the formula: [2] The dry composition for amplifying nucleic acid according to [1] above, wherein the polymer is a homopolymer composed of one type of constitutional unit derived from a monomer represented by formula (1). [3] The polymer has the formula (2):
• the polymer has the formula (3):
• the polymer has the formula (4):
• a reaction solution is prepared by mixing the dry composition for nucleic acid amplification according to any one of [1] to [8] above, a nucleic acid to be amplified, and water; A method for amplifying nucleic acid, comprising carrying out a nucleic acid amplification reaction using the obtained reaction solution.
• the present invention makes it possible to obtain a dry composition for amplifying nucleic acids that has excellent storage stability.
• the present invention provides a dry composition for nucleic acid amplification (sometimes referred to herein as the "dry composition of the present invention") comprising a polymer (sometimes referred to herein as the "polymer of the present invention”) comprising a constitutional unit derived from a monomer represented by formula (1).
• dry composition refers to a solid composition obtained by drying a solution or dispersion.
• dry composition for nucleic acid amplification refers to a dry composition used in a nucleic acid amplification method.
• dry composition for nucleic acid amplification refers to a dry composition for performing a nucleic acid amplification reaction by mixing the dry composition, the nucleic acid to be amplified, and water to prepare a reaction solution and using the resulting reaction solution.
• nucleic acid amplification methods include the Polymerase Chain Reaction (PCR) method, Loop mediated isothermal Amplification (LAMP) method, Transcription Mediated Amplification (TMA) method, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (IICAN) method, Strand Displacement Amplification (SDA) method, Ligase Chain Reaction (LCR) method, and Nucleic Acid Sequence-Based Amplification (NASBA) method.
• PCR Polymerase Chain Reaction
• LAMP Loop mediated isothermal Amplification
• TMA Transcription Mediated Amplification
• IICAN Isothermal and Chimeric primer-initiated Amplification of Nucleic acids
• SDA Strand Displacement Amplification
• LCR Ligase Chain Reaction
• NASBA Nucleic Acid Sequence-Based Amplification
• the nucleic acid amplification method is preferably a polymerase chain reaction (PCR), and more preferably a quantitative polymerase chain reaction. That is, the dry composition of the present invention is more preferably used for a quantitative polymerase chain reaction.
• PCR polymerase chain reaction
• the "polymerase chain reaction (PCR)” includes not only a typical PCR method in which DNA is the target nucleic acid, but also a reverse transcription polymerase chain reaction (RT-PCR) method in which RNA is the target nucleic acid.
• RT-PCR reverse transcription polymerase chain reaction
• the "quantitative polymerase chain reaction” includes not only a typical quantitative polymerase chain reaction method in which DNA is the target nucleic acid, but also a quantitative reverse transcription polymerase chain reaction method in which RNA is the target nucleic acid.
• the polymer of the present invention has the formula (1):
• X1 represents a (meth)acryloyloxy group or a (meth)acryloylamino group
• L 1 represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms
• R 1 to R 3 each independently represent an alkyl group having 1 to 3 carbon atoms.
• the polymer of the present invention may be used alone or in combination of two or more kinds.
• structural unit (1) means a structural unit having a structure formed by reaction of the carbon-carbon double bond of the (meth)acryloyl group contained in monomer (1).
• structural units derived from other monomers have the same meaning as the structural unit derived from monomer (1).
• the monomer (1) may be used alone or in combination of two or more kinds. That is, the polymer of the present invention may be a homopolymer consisting of one kind of structural unit (1), or a copolymer containing two or more kinds of structural units (1).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• X 1 in formula (1) represents a (meth)acryloyloxy group (i.e., CH 2 ⁇ CR-CO-O-, R: hydrogen atom or methyl group) or a (meth)acryloylamino group (i.e., CH 2 ⁇ CR-CO-NH-, R: hydrogen atom or methyl group).
• X 1 is preferably a (meth)acryloyloxy group, more preferably a methacryloyloxy group.
• L 1 represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms.
• the alkylene group may be linear or branched.
• An example of an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group is -C 2 H 4 -.
• An example of an alkyleneoxyalkylene group having 2 to 4 carbon atoms is -C 2 H 4 -O-C 2 H 4 -.
• L 1 is preferably -C 2 H 4 - or -C 2 H 4 -O-C 2 H 4 -, and more preferably -C 2 H 4 - (i.e., an ethylene group).
• R 1 to R 3 each independently represent an alkyl group having 1 to 3 carbon atoms.
• the alkyl group may be linear or branched.
• Examples of the alkyl group having 1 to 3 carbon atoms include a methyl group, an ethyl group, and a propyl group. From the viewpoint of availability of raw materials, R 1 to R 3 are preferably all methyl groups.
• a preferred monomer (1) is one in which X 1 is a (meth)acryloyloxy group, L 1 is -C 2 H 4 - or -C 2 H 4 -O-C 2 H 4 -, and R 1 to R 3 are methyl groups.
• a more preferred monomer (1) is one in which X 1 is a (meth)acryloyloxy group, L 1 is an ethylene group, and R 1 to R 3 are methyl groups (i.e., 2-(meth)acryloyloxyethyl phosphorylcholine).
• a further preferred monomer (1) is 2-methacryloyloxyethyl phosphorylcholine.
• a commercially available product can be used as the monomer (1).
• 2-(meth)acryloyloxyethyl phosphorylcholine basically means “2-acryloyloxyethyl phosphorylcholine or 2-methacryloyloxyethyl phosphorylcholine.” In cases where multiple 2-(meth)acryloyloxyethyl phosphorylcholines may be present, "2-(meth)acryloyloxyethyl phosphorylcholine” means "2-acryloyloxyethyl phosphorylcholine and/or 2-methacryloyloxyethyl phosphorylcholine.” Other terms similar to "2-(meth)acryloyloxyethyl phosphorylcholine” also have the same meaning as "(2-(meth)acryloyloxyethyl phosphorylcholine.”
• polymer composed of structural unit (1) means a polymer in which all structural units (repeating units) in the polymer chain are composed of structural unit (1). Expressions similar to “polymer composed of structural unit (1)” have the same meaning as “polymer composed of structural unit (1)”.
• a polymer composed of structural unit (1) may be a homopolymer composed of one type of structural unit (1) (hereinafter sometimes abbreviated as "homopolymer (1)”) or a copolymer composed of two or more types of structural unit (1), but is preferably a homopolymer (1).
• the weight average molecular weight of the homopolymer (1) is not particularly limited, but is preferably 20,000 to 2,000,000, more preferably 100,000 to 1,500,000, and even more preferably 500,000 to 1,500,000.
• the weight average molecular weight can be determined in terms of polyethylene glycol by gel permeation chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).
• the polymer of the present invention contains, in addition to the structural unit (1), a structural unit of formula (2):
• R 4 represents a hydrogen atom or a methyl group
• R 5 represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms. (hereinafter may be abbreviated as “monomer (2)”).
• the monomer (2) may be used alone or in combination of two or more kinds. That is, the polymer of the present invention may be a copolymer containing one or more kinds of structural units (1) and one or more kinds of structural units (2).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• R4 in formula (2) represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R4 is preferably a methyl group.
• R 5 in formula (2) represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.
• the alkyl group may be linear or branched. It is preferably a hydrogen atom or an alkyl group having 2 to 20 carbon atoms, more preferably a hydrogen atom or an alkyl group having 3 to 19 carbon atoms, and even more preferably a hydrogen atom or a linear alkyl group having 4 to 18 carbon atoms.
• R 5 is preferably a linear alkyl group having 3 to 6 carbon atoms.
• monomer (2) examples include (meth)acrylic acid, 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, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, and stearyl (meth)acrylate.
• Commercially available products can be used as monomer (2).
• One embodiment of the polymer of the present invention is a copolymer consisting of structural units (1) and (2) (hereinafter sometimes abbreviated as "copolymer (1-2)").
• the ratio of structural unit (1) i.e., the ratio of monomer (1) used in polymerization
• a total of 100 moles of structural units (1) and (2) i.e., a total of 100 moles of monomer (1) and monomer (2) used in polymerization
• the ratio of structural unit (2) i.e., the ratio of monomer (2) used in polymerization
• the weight average molecular weight of the copolymer (1-2) is not particularly limited, but is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,500,000, and even more preferably 30,000 to 1,000,000.
• the polymer of the present invention contains, in addition to the structural unit (1), a structural unit of formula (3):
• R 6 represents a hydrogen atom or a methyl group
• R 7 represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups. (hereinafter may be abbreviated as “monomer (3)”).
• the monomer (3) may be used alone or in combination of two or more kinds. That is, the polymer of the present invention may be a copolymer containing one or more kinds of structural units (1) and one or more kinds of structural units (3), or may be a copolymer containing one or more kinds of structural units (1), one or more kinds of structural units (2) and one or more kinds of structural units (3).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• R6 represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R6 is preferably a methyl group.
• R7 represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups.
• the number of hydroxy groups in R7 is preferably 2 to 5.
• the alkyl group may be linear or branched. Examples of the alkyl group having 3 to 6 carbon atoms include a propyl group, a butyl group, a pentyl group, and a hexyl group.
• monomer (3) examples include glycerin mono(meth)acrylate, threitol mono(meth)acrylate, erythritol mono(meth)acrylate, xylitol mono(meth)acrylate, arabitol mono(meth)acrylate, mannitol mono(meth)acrylate, galactitol mono(meth)acrylate, sorbitol mono(meth)acrylate, etc.
• glycerin mono(meth)acrylate and xylitol mono(meth)acrylate are preferred, glycerin mono(meth)acrylate is more preferred, and glycerin monomethacrylate is even more preferred.
• Monomer (3) may be a commercially available product or may be produced by a known method.
• monomer (3) can be produced by an esterification reaction between (meth)acrylic acid or a derivative thereof (e.g., an acid chloride) and a polyhydric alcohol having three or more hydroxyl groups. Esterification reactions are well known, and a person skilled in the art can carry out the reaction by appropriately setting the conditions.
• One embodiment of the polymer of the present invention is a copolymer consisting of structural units (1) and (3) (hereinafter sometimes abbreviated as "copolymer (1-3)").
• the ratio of structural unit (1) i.e., the ratio of monomer (1) used in polymerization
• a total of 100 moles of structural units (1) and (3) i.e., a total of 100 moles of monomer (1) and monomer (3) used in polymerization
• the ratio of structural unit (3) i.e., the ratio of monomer (3) used in polymerization
• the weight average molecular weight of the copolymer (1-3) is not particularly limited, but is preferably 100,000 to 1,000,000, more preferably 100,000 to 800,000, and even more preferably 100,000 to 500,000.
• the polymer of the present invention is a copolymer consisting of structural units (1), (2), and (3) (hereinafter sometimes abbreviated as "copolymer (1-2-3)").
• the ratio of the structural unit (1) i.e., the ratio of the monomer (1) used in the polymerization
• the ratio of the structural unit (1) is preferably 30 to 80 moles, more preferably 30 to 70 moles, and even more preferably 30 to 60 moles
• the ratio of the structural unit (2) i.e., the ratio of the monomer (2) used in the polymerization
• the ratio of the structural unit (3) i.e., the ratio of the monomer (3) used in the polymer
• the weight average molecular weight of the copolymer (1-2-3) is not particularly limited, but is preferably 10,000 to 500,000, more preferably 10,000 to 100,000, and even more preferably 10,000 to 50,000.
• the polymer of the present invention contains, in addition to the structural unit (1), a structural unit of formula (4):
• R8 represents a hydrogen atom or a methyl group
• n represents a number from 1 to 10
• R 9 represents a hydrogen atom, a methyl group, or an ethyl group. (hereinafter may be abbreviated as “monomer (4)”).
• the polymer of the present invention may be a copolymer containing one or more types of structural units (1) and one or more types of structural units (4), or may be a copolymer containing one or more types of structural units (1), one or more types of structural units (4), and one or more other structural units (e.g., one or more types of structural units (2), and/or one or more types of structural units (3)).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• R8 represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R8 is preferably a methyl group.
• n is a number from 1 to 10, and preferably a number from 8 to 10. Note that n represents the number of OCH 2 CH 2 units in the polyethylene glycol chain, and the actual n of the monomer (4) is an average value. Therefore, n may be a decimal number.
• R 9 in formula (4) represents a hydrogen atom, a methyl group, or an ethyl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.
• monomer (4) examples include polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, and ethoxypolyethylene glycol (meth)acrylate. From the viewpoint of storage stability of the polymer, methoxypolyethylene glycol (meth)acrylate and ethoxypolyethylene glycol (meth)acrylate are preferred, and methoxypolyethylene glycol methacrylate is more preferred.
• One embodiment of the polymer of the present invention is a copolymer consisting of structural units (1) and (4) (hereinafter sometimes abbreviated as "copolymer (1-4)").
• the ratio of structural unit (1) i.e., the ratio of monomer (1) used in polymerization
• a total of 100 moles of structural units (1) and (4) i.e., 100 moles of monomer (1) and monomer (4) used in polymerization
• the ratio of structural unit (4) i.e., the ratio of monomer (4) used in polymerization
• the weight average molecular weight of the copolymer (1-4) is not particularly limited, but is preferably 100,000 to 1,000,000, more preferably 200,000 to 1,000,000, and even more preferably 300,000 to 800,000.
• the polymer of the present invention may contain other structural units derived from other monomers different from the monomers (1) to (4) as long as the effect of the present invention is not impaired. Only one type of other monomer may be used, or two or more types may be used in combination. There is no particular limitation on the other monomer, but examples include benzyl (meth)acrylate and isobornyl (meth)acrylate.
• the amount of other structural units in the polymer of the present invention is preferably 20 mol % or less based on the total structural units. It is more preferable that the polymer of the present invention does not contain other structural units.
• the polymer of the present invention is Preferably, it is at least one selected from the group consisting of homopolymer (1), copolymer (1-2), copolymer (1-3), copolymer (1-4), and copolymer (1-2-3), More preferably, it is at least one selected from the group consisting of homopolymer (1), copolymer (1-2), copolymer (1-4), and copolymer (1-2-3), More preferred are the homopolymer (1), the copolymer (1-2), the copolymer (1-3) and the copolymer (1-2-3).
• examples of the monomers (1) to (4) for forming the structural units (1) to (4) include those mentioned above.
• the polymer of the present invention can be produced by known methods (e.g., the method described in WO 2018/216628, etc.).
• the content of the polymer of the present invention in the entire dry composition for nucleic acid amplification of the present invention is preferably 0.01% by mass to 10% by mass, more preferably 0.05% by mass to 5% by mass, and even more preferably 0.1% by mass to 1.0% by mass, from the viewpoint of stabilizing effect.
• the above content means the total content of two or more types of the polymer of the present invention.
• the above content or amount means the total content or amount of the two or more types of the components.
• the dry composition of the present invention contains components other than the polymer of the present invention (sometimes referred to as "other components" in this specification).
• other components known components used in nucleic acid amplification methods such as PCR can be used.
• polymerase, primer, substrate, fluorescent DNA staining reagent, fluorescent probe, passive reference, salt, surfactant, protein, nucleic acid, etc. can be mentioned.
• only one kind may be used, or two or more kinds may be used in combination.
• DNA polymerase known DNA polymerases can be used. From the viewpoint of heat resistance, enzymes derived from thermophilic bacteria, thermophilic archaea, hyperthermophilic bacteria, and hyperthermophilic archaea, and mutant enzymes thereof are preferred.
• the DNA polymerase is appropriately selected from DNA-dependent DNA polymerases, RNA-dependent DNA polymerases, or enzymes having both functions, depending on the purpose of nucleic acid amplification. In addition, whether to use a DNA polymerase having nuclease activity or a DNA polymerase without nuclease activity is appropriately selected.
• the dry composition of the present invention preferably contains a polymerase.
• the content i.e., the amount (U) of polymerase per 1 mg of the dry composition
• U the amount of polymerase per 1 mg of the dry composition
• the primer is not particularly limited, but examples include oligonucleotides of about 15 to 30 bases designed and prepared by known methods.
• the primer may be appropriately modified with a fluorescent dye such as fluorescein (FAM). Only one type of primer may be used, or two types of primers may be used as a pair, or multiple primers may be used to amplify multiple regions simultaneously.
• FAM fluorescein
• the dry composition of the present invention preferably contains a primer.
• the content thereof i.e., the amount of primer (pmol) per 1 mg of the dry composition
• the content thereof is preferably 1.0 to 10.0 pmol/mg, more preferably 2.0 to 8.0 pmol/mg, and even more preferably 3.0 to 6.0 pmol/mg.
• the substrate is not particularly limited, but may be, for example, a mixture of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP) (dNTPs).
• dATP deoxyadenosine triphosphate
• dTTP deoxythymidine triphosphate
• dGTP deoxyguanosine triphosphate
• dCTP deoxycytidine triphosphate
• the fluorescent DNA staining reagent is not particularly limited, but may be, for example, SYBRTM Green I.
• the fluorescent probe is not particularly limited, but may be, for example, a TaqMan TM probe.
• the passive reference may be appropriately selected depending on the purpose of nucleic acid amplification. Examples of the passive reference include ROX TM Dye.
• Salts are not particularly limited, but examples include salts of organic bases such as tris(hydroxymethyl)aminomethane, tricine, and bicine with acids such as sulfuric acid, hydrochloric acid, acetic acid, and phosphoric acid.
• magnesium salts e.g., magnesium chloride
• manganese salts e.g., manganese acetate
• potassium chloride ammonium sulfate, and the like may also be used as salts.
• the surfactant is not particularly limited, but examples thereof include polyoxyethylene sorbitan fatty acid esters and polyoxyethylene alkylphenyl ethers.
• the protein is not particularly limited, but may be, for example, bovine serum albumin.
• any DNA and/or RNA may be used, for example as an exogenous control gene.
• the nucleic acid may be synthesized in vitro, 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, plants, etc., or may be isolated and cultured.
• the dry composition of the present invention may further contain an oil such as mineral oil; or a solid phase carrier such as glass beads or magnetic beads.
• kit-type products that combine multiple selected components of the above, as well as master mix-type products (sometimes called primer mixes, premixes, etc.) in which these components are premixed, may also be used.
• master mix-type products sometimes called primer mixes, premixes, etc.
• the dry composition of the present invention can be produced, for example, by drying a solution containing the polymer of the present invention, other components (particularly, polymerase and/or primer), and water.
• drying methods for this purpose include a method of drying at room temperature and atmospheric pressure (air drying method), a method of drying at room temperature or higher and at a pressure of 90% or less of atmospheric pressure (evaporation drying method), and a method of freezing a solution and drying it at a low temperature and under high vacuum (freeze drying method).
• the drying method is preferably the freeze drying method.
• the solution for producing the dry composition of the present invention may contain a buffer.
• the buffer is not particularly limited, but may be, for example, a mixture of an organic base such as tris(hydroxymethyl)aminomethane, tricine, or bicine with an acid such as sulfuric acid, hydrochloric acid, acetic acid, or phosphoric acid to adjust the pH to 6 to 9, more preferably 7 to 8.
• the buffer desirably contains a magnesium salt (e.g., magnesium chloride) and/or a manganese salt (e.g., manganese acetate) as appropriate.
• the buffer may further contain a salt such as potassium chloride or ammonium sulfate.
• the buffer may further contain a water-soluble organic solvent such as dimethyl sulfoxide, dimethylformamide, formamide, or glycerin.
• the buffer may further contain a surfactant such as polyoxyethylene sorbitan fatty acid ester or polyoxyethylene alkylphenyl ether.
• the buffer may further contain a protein such as bovine serum albumin.
• the present invention also provides a method for amplifying nucleic acid, comprising mixing the dry composition of the present invention, a nucleic acid to be amplified, and water to prepare a reaction solution, and carrying out a nucleic acid amplification reaction using the reaction solution obtained.
• the reaction solution may be prepared, for example, by mixing a sample containing the nucleic acid to be amplified and water (i.e., an aqueous nucleic acid solution) with the dry composition of the present invention.
• the nucleic acid to be amplified can be either DNA or RNA.
• the nucleic acid amplification method is as described above.
• the nucleic acid amplification method of the present invention is preferably a polymerase chain reaction (PCR), more preferably a quantitative polymerase chain reaction.
• PCR polymerase chain reaction
• the nucleic acid amplification method (particularly the PCR) is well known to those skilled in the art, and can be appropriately performed by those skilled in the art.
• polymer 1 The weight average molecular weight of polymer 1 was 1,030,000 in terms of polyethylene glycol, as measured by gel permeation chromatography (hereinafter referred to as "GPC") under the conditions described below.
• polymer 2 a copolymer (hereinafter referred to as "polymer 2").
• the weight average molecular weight of polymer 2 was 22,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• polymer 4 a copolymer (hereinafter referred to as "polymer 4").
• the weight average molecular weight of polymer 4 was 501,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• nucleic acid to be amplified is double-stranded DNA and the nucleic acid amplification method is the PCR method.
• Solution A was prepared by mixing the components shown in Table 2. Note that the preparation of solution A was carried out while cooling with ice.
• compositions for nucleic acid amplification Preparation of the composition for nucleic acid amplification before drying
• the polymers obtained in Synthesis Examples 1 to 4 were added to the obtained solution A to prepare compositions for nucleic acid amplification.
• 2 ⁇ L of an aqueous solution of polymer 1, 2, or 4 having a concentration of 0.5 w/v% was added to solution A per well to prepare 15 ⁇ L of a composition for nucleic acid amplification (polymer concentration in each composition: 0.067 w/v%).
• Example 3 2 ⁇ L of an aqueous solution of polymer 3 having a concentration of 0.05 w/v% was added to solution A per well to prepare 15 ⁇ L of a composition for nucleic acid amplification (polymer concentration in the composition: 0.0067 w/v%). Note that the preparation of these compositions was carried out while cooling with ice.
• ⁇ Preparation of dry composition for nucleic acid amplification> The obtained composition for nucleic acid amplification was placed in a PCR tube (manufactured by Eppendorf), and the tube was placed in an ultra-low temperature refrigerator (freeze ultra-low temperature bath manufactured by Nippon Freezer Co., Ltd.) and stored at -80°C for 30 minutes to freeze the composition.
• the PCR tube containing the frozen composition was placed in a freeze dryer ("FDU-2100" manufactured by Tokyo Rikakikai Co., Ltd.) to freeze-dry (sublimation drying) the composition to prepare a dried composition for nucleic acid amplification.
• the freeze-drying was performed at a reduced pressure of 15 kPa to 20 kPa and at a temperature of 22°C for 24 hours.
• the content of each polymer in the obtained dried composition was 0.4% by mass
• the content of polymerase was 0.2 U/mg
• the content of primer i.e., the total content of the first and second primers
• the band intensity of the nucleic acid amplification product was measured as follows using the dried composition immediately after freeze-drying or after three months of storage.
• the dry composition was stored as follows. Specifically, after freeze-drying, the PCR tube was removed from the freeze-dryer and immediately sealed with parafilm to prevent the dry composition from absorbing moisture. The sealed PCR tube containing the dry composition was stored at 37° C. for 3 months.
• PCR reaction solution A target nucleic acid solution with a nucleic acid concentration of 10 ng/ ⁇ L was added to the composition for nucleic acid amplification in an amount of 5 ⁇ L per well to prepare a PCR reaction solution (polymer concentration in each PCR reaction solution: 0.05 w/v% in Examples 1, 2, and 4, and 0.005 w/v% in Example 3). Note that the PCR reaction solutions were all prepared while being cooled on ice.
• PCR was performed using the PCR reaction solution obtained as described above with a qPCR device (Applied Biosystems'"StepOnePlus TM Real-Time PCR System") under the reaction conditions (temperature program) shown in Table 3 below.
• ⁇ Electrophoresis> The PCR product obtained was electrophoresed, and the DNA amount (band density) was observed as follows. First, 4 ⁇ L of 6x Loading Buffer (manufactured by Nippon Gene Co., Ltd.) was added to 20 ⁇ L of the obtained PCR product to prepare an electrophoretic sample. A gel for electrophoresis was prepared using Agarose S (manufactured by Nippon Gene Co., Ltd.). 1x TEA (manufactured by Nippon Gene Co., Ltd.) was used as the electrophoretic buffer.
• 6x Loading Buffer manufactured by Nippon Gene Co., Ltd.
• a marker (Gene Ladder 100 (0.1-2 kbp), manufactured by Nippon Gene Co., Ltd.) and an electrophoretic sample were added to the prepared electrophoretic gel at 4 ⁇ L/well, and the mixture was electrophoresed at 100 V for 30 minutes. Thereafter, the sample components were stained using Midori Green Advance (Genetics), then destained using Distilled Water, Nuclease-free (Nippon Gene Co., Ltd.), and the band of the target nucleic acid amplification product (size: 1.3 kbp) was detected using a gel imaging device (Fas-Digi Compact, Nippon Genetics Co., Ltd.).
• Comparative Example 1 In Comparative Example 1, the same operations as in Examples 1 to 4 were carried out, except that in ⁇ Preparation of dry composition for nucleic acid amplification>, 2 ⁇ L of distilled water, nuclease-free (manufactured by Nippon Gene Co., Ltd.) was used instead of 2 ⁇ L of the aqueous solution of the polymer.
• the band intensities (relative values) obtained using the dry composition after storage for three months, with the band intensity obtained using the dry composition immediately after freeze-drying taken as 100%, are shown in Table 4 below. Also, FIG. 1 shows the results of electrophoresis performed using the dry composition after storage for three months.
• nucleic acid to be amplified is single-stranded RNA and the nucleic acid amplification method is the RT-qPCR method.
• composition for nucleic acid amplification Preparation of the composition for nucleic acid amplification before drying
• the polymer obtained in Synthesis Example 1 or 3 to 5 was added to the obtained solution B to prepare a composition for nucleic acid amplification.
• 2 ⁇ L of an aqueous solution of polymer 1, 4, or 5 having a concentration of 0.5 w/v% was added to solution B per well to prepare 15 ⁇ L of a composition for nucleic acid amplification (concentration of polymer in each composition: 0.067 w/v%).
• Example 6 2 ⁇ L of an aqueous solution of polymer 3 having a concentration of 0.05 w/v% was added to solution B per well to prepare 15 ⁇ L of a composition for nucleic acid amplification (concentration of polymer in composition: 0.0067 w/v%). Note that the preparation of these compositions was carried out while cooling with ice.
• ⁇ Preparation of dry composition for nucleic acid amplification> The obtained composition for nucleic acid amplification was placed in a PCR tube (manufactured by Eppendorf), and the tube was placed in an ultra-low temperature refrigerator (freeze ultra-low temperature bath manufactured by Nippon Freezer Co., Ltd.) and stored at -80°C for 30 minutes to freeze the composition.
• the PCR tube containing the frozen composition was placed in a freeze dryer ("FDU-2100" manufactured by Tokyo Rikakikai Co., Ltd.) to freeze-dry (sublimation drying) the composition to prepare a dried composition for nucleic acid amplification.
• the freeze-drying was performed at a reduced pressure of 15 kPa to 20 kPa and at a temperature of 22°C for 24 hours.
• the content of each polymer in the obtained dried composition was 0.4% by mass, and the content of the primer (i.e., the total content of the forward primer and the reverse primer) was 4.0 pmol/mg.
• the dried composition immediately after freeze-drying or the dried composition after 12 days of storage was used to measure the fluorescence intensity of the nucleic acid amplification product as follows.
• the dry composition was stored as follows. Specifically, after freeze-drying, the PCR tube was removed from the freeze-dryer and immediately sealed with parafilm to prevent the dry composition from absorbing moisture. The sealed PCR tube containing the dry composition was stored at 60° C. for 12 days.
• RT-qPCR reaction solution An aqueous solution of target nucleic acid with a nucleic acid concentration of 100 copies/ ⁇ L was added to the composition for nucleic acid amplification in an amount of 5 ⁇ L per well to prepare an RT-qPCR reaction solution (polymer concentration in each RT-qPCR reaction solution: 0.05 w/v% in Examples 5, 7, and 8, and 0.005 w/v% in Example 6). Note that the PCR reaction solutions were all prepared while being cooled on ice.
• RT-qPCR was performed using the RT-qPCR reaction solution obtained as described above with a qPCR device (Applied Biosystems'"StepOnePlus TM Real-Time PCR System"), and the fluorescence intensity at the end point of the RT-qPCR reaction solution was measured.
• the reaction conditions are as shown in Table 6 below.
• the fluorescence intensity (relative value) obtained using the dried composition after 12 days of storage is shown in Table 7 below, assuming that the fluorescence intensity obtained using the dried composition immediately after freeze-drying is 100%.
• Comparative Example 2 In Comparative Example 2, the same operations as in Examples 5 to 8 were carried out, except that in ⁇ Preparation of dry composition for nucleic acid amplification>, 2 ⁇ L of distilled water, nuclease-free (manufactured by Nippon Gene Co., Ltd.) was used instead of 2 ⁇ L of the aqueous solution of the polymer.
• the fluorescence intensities (relative values) obtained using the dry composition after 12 days of storage are shown in Table 7 below, assuming that the fluorescence intensity obtained using the dry composition immediately after lyophilization is 100%.
• the dry compositions of Examples 5 to 8 containing polymers 1 or 3 to 5 after 12 days of storage had higher fluorescence intensity (relative value) than the dry composition of Comparative Example 2 which did not contain these polymers.
• the fluorescence intensity (relative value) obtained using the dry composition after 12 days of storage was 82%.
• the dry composition of the present invention is useful for nucleic acid amplification methods (particularly quantitative polymerase chain reaction) for genetic testing, microbiological testing, viral testing, etc.

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