Classifications

• • 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
• • 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
• • 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 freeze-thaw protection agent for a composition for nucleic acid amplification and a method for amplifying nucleic acid 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.
• nucleic acid amplification reagents such as oligonucleotides called primers and enzymes.
• These nucleic acid amplification reagents are unstable at room temperature or under refrigerated conditions, so they are stored under frozen conditions (for example, -80°C to -20°C) and thawed when used, which involves repeated freezing and thawing.
• freezing and thawing proteins changes their higher-order structure and causes them to become inactivated.
• Nucleic acid amplification compositions to which sugars, glycerin, albumin, DMSO, etc. have been added as freeze-thaw protection agents are less susceptible to inactivation due to freeze-thawing than compositions to which no such agents have been added.
• problems with the sugars, etc. including the need to add them in high concentrations, the need to remove them before use, and the fact that they affect the properties of the target substance.
• the present invention has been made in light of the above-mentioned circumstances, and its purpose is to provide a freeze-thaw protectant for a nucleic acid amplification composition that can reduce the inactivation of the nucleic acid amplification composition due to freeze-thawing at a relatively low concentration.
• 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.
• a freeze-thaw protection agent for a composition for amplifying nucleic acid which is a polymer containing a structural unit derived from a monomer represented by the following formula:
• the polymer has the formula (2):
• R 4 represents a hydrogen atom or a methyl group
• R 5 represents a hydrogen atom, an alkyl group having 1 to 20 carbon atoms, a -(OCH 2 CH 2 ) n OR 6 group (wherein n represents a number from 1 to 10, and R 6 represents a hydrogen atom, a methyl group, or an ethyl group), or an aralkyl group having 7 to 12 carbon atoms.
• the freeze-thaw protectant for the composition for amplifying nucleic acid according to any one of [4] to [6] above, wherein the weight-average molecular weight of the copolymer is 10,000 to 2,000,000, preferably 20,000 to 1,500,000, and more preferably 30,000 to 1,000,000.
• the freeze-thaw protectant for the composition for amplifying nucleic acid according to any one of [8] to [10] above, wherein the weight-average molecular weight of the copolymer is 100,000 to 1,000,000, preferably 200,000 to 1,000,000, and more preferably 300,000 to 800,000.
• the freeze-thaw protectant for the composition for amplifying nucleic acid according to any one of [12] to [14] above, wherein the weight-average molecular weight of the copolymer is 50,000 to 1,000,000, preferably 100,000 to 800,000, and more preferably 100,000 to 500,000.
• a composition for nucleic acid amplification comprising the freeze-thaw protection agent of the composition for nucleic acid amplification according to any one of [1] to [15] above, and water.
• composition for nucleic acid amplification according to any one of [16] to [18] above, wherein the concentration of the freeze-thaw protection agent in the composition for nucleic acid amplification is 0.00001 to 10 w/v %, preferably 0.001 to 1 w/v %, and more preferably 0.01 to 0.5 w/v %.
• concentration of the freeze-thaw protection agent in the composition for nucleic acid amplification is 0.00001 to 10 w/v %, preferably 0.001 to 1 w/v %, and more preferably 0.01 to 0.5 w/v %.
• a method for amplifying nucleic acid comprising mixing the composition for amplifying nucleic acid according to any one of [16] to [19] above with a sample containing a nucleic acid to be amplified to prepare a reaction solution for amplifying nucleic acid.
• a method for amplifying nucleic acid comprising mixing a composition for nucleic acid amplification obtained by thawing the frozen composition for nucleic acid amplification described in [20] above with a sample containing a nucleic acid to be amplified to prepare a reaction solution for nucleic acid amplification.
• the freeze-thaw protection agent of the nucleic acid amplification composition of the present invention can be added to the nucleic acid amplification composition to reduce inactivation of the nucleic acid amplification composition due to freeze-thawing.
• the present invention provides a freeze-thaw protection agent for a composition for nucleic acid amplification (hereinafter may be referred to as the "protection agent of the present invention"), which is a polymer (hereinafter may be referred to as the "polymer of the present invention”) containing a constituent unit derived from a monomer represented by formula (1).
• composition for nucleic acid amplification refers to a composition used in a nucleic acid amplification method.
• freeze-thaw protection agent for a composition for nucleic acid amplification refers to an additive used to reduce inactivation of a composition for nucleic acid amplification due to freeze-thawing.
• 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 includes a structural unit derived from a monomer represented by the following formula: The polymer of the present invention may be used alone or in combination of two or more kinds.
• the "monomer represented by formula (1)” may be abbreviated as “monomer (1)”.
• the “monomers represented by other formulae” may also be abbreviated as “monomer represented by formula (1)” in the same manner.
• the "structural unit derived from monomer (1)” may be abbreviated as “structural unit (1).”
• the "structural unit derived from another monomer” may also be abbreviated as the “structural unit derived from monomer (1).”
• the constituent unit derived from monomer (1) means a constituent unit having a structure formed by reaction of the carbon-carbon double bond of the (meth)acryloyl group contained in monomer (1).
• the constituent units derived from other monomers have the same meaning as the constituent 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 (1) a polymer consisting of structural unit (1) (hereinafter sometimes abbreviated as "polymer (1)").
• polymer consisting of structural unit (1) means a polymer in which all structural units (repeating units) in the polymer chain consist of structural unit (1). Expressions similar to “polymer consisting of structural unit (1)” also have the same meaning as “polymer consisting of structural unit (1)”.
• Polymer (1) may be a homopolymer consisting of one type of structural unit (1) or a copolymer consisting of two or more types of structural units (1), but is preferably a copolymer consisting of two or more types of structural units (1).
• the weight-average molecular weight of polymer (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 filtration 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, an alkyl group having 1 to 20 carbon atoms, a -(OCH 2 CH 2 ) n OR 6 group (wherein n represents a number from 1 to 10, and R 6 represents a hydrogen atom, a methyl group, or an ethyl group), or an aralkyl group having 7 to 12 carbon atoms.
• 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 represents a hydrogen atom, an alkyl group having 1 to 20 carbon atoms, a —(OCH 2 CH 2 ) n OR 6 group, or an aralkyl group having 7 to 12 carbon atoms.
• R 5 in formula (2) is a hydrogen atom or an alkyl group having 1 to 20 carbon atoms (hereinafter, sometimes abbreviated as "monomer (2a)”) will be described.
• the alkyl group may be linear or branched.
• R 5 is preferably a hydrogen atom or an alkyl group having 10 to 20 carbon atoms, more preferably a hydrogen atom or an alkyl group having 15 to 19 carbon atoms, and even more preferably a hydrogen atom or a linear alkyl group having 17 to 19 carbon atoms.
• monomer (2a) 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. Only one type of monomer (2a) may be used, or two or more types may be used in combination. Commercially available products
• (i) (meth)acrylic acid, 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 are preferred;
• (ii) (meth)acrylic acid, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, and stearyl (meth)acrylate are more preferred;
• (iii) (meth)acrylic acid and stearyl (meth)acrylate are more preferred;
• Methacrylic acid and stearyl methacrylate are particularly preferred.
• One embodiment of the polymer of the present invention is a copolymer consisting of structural unit (1) and structural unit (2a) (hereinafter sometimes abbreviated as "copolymer (1-2a)").
• the monomer (1) and the monomer (2a) may each be used alone or in combination of two or more kinds. That is, the copolymer (1-2a) may be a copolymer containing one or more kinds of structural unit (1) and one or more kinds of structural unit (2a).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• the ratio of the structural unit (1) i.e., the ratio of the monomer (1) used in the polymerization
• a total of 100 moles of the structural unit (1) and the structural unit (2a) i.e., a total of 100 moles of the monomer (1) and the monomer (2a) used in the polymerization
• the ratio of the structural unit (2a) i.e., the ratio of the monomer (2a) used in the polymerization
• the weight-average molecular weight of the copolymer (1-2a) 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 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).
• R 5 in formula (2) is a -(OCH 2 CH 2 ) n OR 6 group (wherein n is a number from 1 to 10, and R 6 is a hydrogen atom, a methyl group, or an ethyl group) (hereinafter sometimes abbreviated as "monomer (2b)") will be described.
• R6 is preferably a methyl group or an ethyl group, more preferably a methyl group.
• n is a number from 1 to 10, preferably a number from 5 to 10, and more preferably a number from 8 to 10.
• monomer (2b) examples include polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, and ethoxypolyethylene glycol (meth)acrylate. Only one type of monomer (2b) may be used, or two or more types may be used in combination. 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. Commercially available products can be used as monomer (2b).
• One embodiment of the polymer of the present invention is a copolymer consisting of structural unit (1) and structural unit (2b) (hereinafter sometimes abbreviated as "copolymer (1-2b)").
• the monomer (1) and the monomer (2b) may each be used alone or in combination of two or more kinds. That is, the copolymer (1-2b) may be a copolymer containing one or more kinds of structural unit (1) and one or more kinds of structural unit (2b).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• the ratio of the structural unit (1) i.e., the ratio of the monomer (1) used in the polymerization
• a total of 100 moles of the structural unit (1) and the structural unit (2b) i.e., a total of 100 moles of the monomer (1) and the monomer (2b) used in the polymerization
• the ratio of the structural unit (2b) i.e., the ratio of the monomer (2b) used in the polymerization
• the weight average molecular weight of copolymer (1-2b) 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.
• R 5 in formula (2) is an aralkyl group having 7 to 12 carbon atoms (hereinafter, may be abbreviated as "monomer (2c)”) will be described.
• the alkyl group in the aralkyl group having 7 to 12 carbon atoms may be linear or branched.
• Examples of the aralkyl group having 7 to 12 carbon atoms include a benzyl group, a 2-phenylethyl group, a 3-phenylpropyl group, a 4-phenylbutyl group, and a 5-phenylpentyl group.
• the number of carbon atoms in the aralkyl group is more preferably 7 to 9.
• monomer (2c) examples include benzyl (meth)acrylate, 2-phenylethyl (meth)acrylate, 3-phenylpropyl (meth)acrylate, 4-phenylbutyl (meth)acrylate, and 5-phenylpentyl (meth)acrylate. From the viewpoint of storage stability of the polymer, benzyl (meth)acrylate, 2-phenylethyl (meth)acrylate, and 3-phenylpropyl (meth)acrylate are preferred, and benzyl methacrylate is more preferred. Only one type of monomer (2c) may be used, or two or more types may be used in combination. Commercially available products can be used as monomer (2c).
• One embodiment of the polymer of the present invention is a copolymer consisting of structural unit (1) and structural unit (2c) (hereinafter sometimes abbreviated as "copolymer (1-2c)").
• the monomer (1) and the monomer (2c) may each be used alone or in combination of two or more kinds. That is, the copolymer (1-2c) may be a copolymer containing one or more kinds of structural unit (1) and one or more kinds of structural unit (2c).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• the ratio of structural unit (1) i.e., the ratio of monomer (1) used in polymerization
• structural unit (2c) i.e., a total of 100 moles of monomer (1) and monomer (2c) used in polymerization
• the ratio of structural unit (2c) is preferably 5 to 60 moles, more preferably 5 to 40 moles, and even more preferably 10 to 30 moles.
• the weight average molecular weight of the copolymer (1-2c) is not particularly limited, but is preferably 50,000 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 may contain other structural units derived from other monomers different from monomer (1) and monomer (2) (i.e., monomers (2a) to (2c)) to the extent that 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 glycerol methacrylate 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 can be produced by known methods (e.g., the method described in WO 2018/216628, etc.).
• the amount of the protecting agent of the present invention (i.e., the polymer of the present invention) used is determined by its concentration in the composition for amplifying nucleic acid, which will be described later. From the viewpoint of protective effect, the concentration of the protecting agent of the present invention in the composition is preferably 0.00001 to 10 w/v%, more preferably 0.001 to 1 w/v%, and even more preferably 0.01 to 0.5 w/v%.
• the above concentration means the total concentration of the two or more types of protecting agents.
• the above concentration also means the total concentration of the two or more types of the components.
• the present invention provides a composition for amplifying nucleic acid comprising the protecting agent of the present invention and water (hereinafter, may be referred to as "the composition of the present invention”).
• the protecting agent of the present invention is as described above.
• nucleic acid amplification methods examples 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 (ICAN) 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
• ICAN 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 composition of the present invention is more preferably used in a quantitative polymerase chain reaction.
• PCR polymerase chain reaction
• RT-PCR reverse transcription polymerase chain reaction
• 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 composition of the present invention contains components other than the polymer of the present invention (hereinafter, sometimes referred to as "other components").
• other components known components used in nucleic acid amplification methods, such as PCR, can be used.
• polymerase, primer, buffer, substrate, 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 composition of the present invention preferably contains a polymerase.
• the content thereof i.e., the amount (U) of polymerase per 1 ⁇ L of the composition of the present invention
• U the amount of polymerase per 1 ⁇ L of the composition of the present invention
• 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 composition of the present invention preferably contains a primer.
• the content thereof i.e., the amount of primer (pmol) per 1 ⁇ L of the composition of the present invention
• the content thereof is preferably 0.1 to 1 pmol/ ⁇ L.
• the buffer is not particularly limited, but examples thereof include 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, and adjusted to a pH of 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 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 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 from cells, microorganisms, viruses, etc. by known methods.
• 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.
• composition of the present invention may further contain a solid phase carrier such as an oil, such as mineral oil, glass beads, or magnetic beads.
• a solid phase carrier such as an oil, such as mineral oil, 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.
• composition of the present invention contains the protective agent of the present invention, inactivation due to freezing and thawing is suppressed. Therefore, the composition of the present invention is preferably stored frozen. This frozen composition of the present invention is also within the scope of the present invention.
• the present invention also provides a nucleic acid amplification method comprising mixing the composition of the present invention with a sample containing a nucleic acid to be amplified to prepare a reaction solution for nucleic acid amplification.
• the present invention also provides a nucleic acid amplification method comprising mixing a composition for nucleic acid amplification obtained by thawing a frozen composition of the present invention (hereinafter, sometimes referred to as a "composition after freezing and thawing”) with a sample containing a nucleic acid to be amplified to prepare a reaction solution for nucleic acid amplification.
• the reaction solution can be produced, for example, by mixing a sample containing a nucleic acid to be amplified and water (i.e., an aqueous nucleic acid solution) with the composition of the present invention or the composition after freezing and thawing.
• a sample containing a nucleic acid to be amplified and water i.e., an aqueous nucleic acid solution
• the composition of the present invention in the nucleic acid amplification method of the present invention is as described above.
• 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 (qPCR).
• PCR polymerase chain reaction
• qPCR quantitative 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 After sufficient nitrogen replacement in the reaction vessel, polymerization was carried out by heating at 70°C for 6 hours under stirring. The resulting reaction solution was ice-cooled and dropped into acetone to precipitate a polymer. The precipitate was filtered off, washed with acetone, and then vacuum-dried to obtain a white powdery homopolymer (hereinafter referred to as "polymer 1"). The weight average molecular weight of Polymer 1 was 680,000 in terms of polyethylene glycol as measured by 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 501,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.
• nucleic acid to be amplified is single-stranded RNA and the nucleic acid amplification method is the RT-qPCR 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 amplifying nucleic acid were prepared. Specifically, 2 ⁇ L of aqueous solutions of polymers 1 to 4, each having a concentration of 0.5 w/v%, were added to solution A per well to prepare 15 ⁇ L of compositions for nucleic acid amplification (polymer concentration in each composition: 0.067 w/v%). All of these compositions were prepared while cooling on ice.
• 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 refrigerator (manufactured by Sanyo Electric Co., Ltd.) and stored at 4°C for 2 hours to thaw the composition. This freezing and thawing procedure was repeated five times.
• the fluorescence intensity of the nucleic acid amplification product was measured as follows using the composition immediately after preparation or after five freeze-thaw cycles.
• RT-qPCR reaction solution An aqueous solution of the 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%). 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 3 below.
• the protective agent of the present invention can reduce the inactivation of a composition for nucleic acid amplification caused by freezing and thawing.
• a composition for nucleic acid amplification containing the protective agent of the present invention is useful for nucleic acid amplification methods (particularly quantitative polymerase chain reaction) for genetic testing, microbial testing, viral testing, etc.

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• 2024
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