JP2016192941A - Probe for detecting cyp2c9*3 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform discrimination of homo and hetero and discrimination of a wild-type allele highly accurately to variant-type allele CYP2C9*3 in a CYP2C9 gene.SOLUTION: A probe includes an oligonucleotide having the entire base length of 15 to 27 which consists of a sequence: 5'-GAGATACMTTGACCT-3'(sequence number 1, M is A or C) or a sequence in which 1 to 6 bases are added to both ends of the sequence, a complementary sequence to the sequence, or a sequence in which 1 to 6 bases are added to both ends of the sequence or the complementary sequence.SELECTED DRAWING: None

Description

  The present invention relates to a CYP2C9 * 3 detection probe for determining a mutant allele (CYP2C9 * 3) present in a cytochrome P450 2C9 (CYP2C9) gene involved in a drug-metabolizing enzyme.

  Cytochrome P450 (CYP) is an enzyme involved in drug metabolism. More than 20 molecular species have been reported for cytochrome P450, followed by “CYP” followed by an Arabic numeral indicating a family, an alphabet indicating a subfamily, and an Arabic numeral indicating a molecular species number. Among them, CYP2C9 is known to be involved in drug metabolism such as ibuprofen, diclofenac, phenytoin, warfarin, tolbutamide, losartan, sulfamethoxazole and many nonsteroidal anti-inflammatory analgesics.

  In addition, mutations in the CYP2C9 gene have identified gene mutations that are associated with reduced drug metabolic activity. In particular, it is known that when 359th isoleucine in the amino acid sequence of CYP2C9 is substituted with leucine, the metabolic activity of warfarin S form is reduced. This substitution mutation is caused by a single nucleotide polymorphism in which the 1075th adenine in the CYP2C9 gene is mutated to cytosine. The rs number given to the single nucleotide polymorphism with this substitution mutation is rs1057910, the mutation allele in the polymorphism is denoted as CYP2C9 * 3, and the wild type allele is denoted as CYP2C9 * 1.

  That is, an individual having a mutant allele CYP2C9 * 3 as a homozygote and an individual having a heterozygote have heterogeneous drug metabolism as compared to a wild-type individual. For this reason, individuals having CYP2C9 * 3 as homozygous and heterozygous individuals have smaller drug doses than wild type individuals. Thus, an appropriate drug dose can be determined by examining the presence or absence of CYP2C9 * 3 before administration for a drug administration subject.

  For example, Patent Document 1 discloses two types of primer sets for analyzing CYP2C9 * 3 in the CYP2C9 gene and a predetermined polymorphism in the VKORC1 gene. Specifically, Patent Document 1 describes that a predetermined region is amplified with a primer set designed to sandwich CYP2C9 * 3, and a polymorphism contained in the amplified fragment is detected with a probe. In Patent Document 1, as a polymorphism detection probe, a probe having a length of 17 to 22 bases including a polymorphic site is disclosed.

  Patent Document 2 discloses that in order to determine the optimal dose of warfarin, the haplotype of the VKORC1 gene is detected and the optimal dose is determined based on this. Patent Document 2 also discloses detecting a haplotype of the VKORC1 gene and detecting a polymorphism of the CYP2C9 gene in Patent Document 2.

Patent No. 5224526 Special table 2008-516630

  However, conventionally, there has not been known a technique capable of highly accurately discriminating homozygous and heterozygous and wild-type alleles of the mutant allele CYP2C9 * 3 in the CYP2C9 gene. For example, Patent Document 1 discloses a probe for detecting a mutant allele CYP2C9 * 3 contained in a fragment amplified with a specific primer set, but the detection sensitivity is not sufficient.

  Therefore, in view of the above situation, the present invention provides a CYP2C9 * 3 detection probe capable of performing homozygous and heterozygous discrimination and wild-type allele discrimination with high accuracy for the mutant allele CYP2C9 * 3 in the CYP2C9 gene. An object of the present invention is to provide a data acquisition method for predicting the drug metabolic ability used.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have found an appropriate base sequence capable of detecting the mutant allele CYP2C9 * 3 in the CYP2C9 gene, and have completed the present invention. In addition, the probe for determining the genotype of the polymorphism cannot be produced arbitrarily as long as the location of the gene polymorphism is elucidated, and it is necessary to find a probe having an appropriate base sequence for each mutation of the polymorphism. is there.

(1) Sequence: 5′-GAGATACMTTGACCT-3 ′ (SEQ ID NO: 1, M is A or C) or a sequence complementary to the sequence, or 1 to 6 at both ends of the sequence or the complementary sequence A probe for CYP2C9 * 3 detection, comprising an oligonucleotide consisting of a sequence to which a base is added and having a total length of 15 to 27 bases.
(2) The base sequence of the oligonucleotide is A, 5'-CA-3 ', 5'-CCA-3', 5'-TCCA-3 ', 5 at the 5' end of the base sequence shown in SEQ ID NO: 1. Either '-GTCCA-3' or 5'-GGTCCA-3 'is added, and T, 5'-TC-3', 5'-TCT-3 ', 5'-TCTC is added to the 3' end of the above sequence. The base sequence to which any of -3 ′, 5′-TCTCC-3 ′ and 5′-TCTCCC-3 ′ is added or a complementary sequence to the base sequence is described in (1) CYP2C9 * 3 detection probe.
(3) The CYP2C9 * 3 detection probe according to (1), wherein the base sequence of the oligonucleotide is any one of SEQ ID NOs: 2 to 7 or a complementary sequence to the base sequence .
AGAGATACMTTGACCTT (SEQ ID NO: 2)
CAGAGATACMTTGACCTTC (SEQ ID NO: 3)
CCAGAGATACMTTGACCTTCT (SEQ ID NO: 4)
TCCAGAGATACMTTGACCTTCTC (SEQ ID NO: 5)
GTCCAGAGATACMTTGACCTTCTCC (SEQ ID NO: 6)
GGTCCAGAGATACMTTGACCTTCTCCC (SEQ ID NO: 7)
(4) A microarray having the CYP2C9 * 3 detection probe according to any one of (1) to (3) above.
(5) amplifying a nucleic acid fragment containing a polymorphic site specified by rs1057910 in the CYP2C9 gene using a subject-derived genome as a template;
Contacting the obtained nucleic acid fragment with the CYP2C9 * 3 detection probe according to any one of (1) to (3) above,
A step of detecting hybridization between the obtained nucleic acid fragment and the CYP2C9 * 3 detection probe,
A data acquisition method for predicting drug metabolizing ability to determine the genotype of rs1057910.
(6) The data acquisition method for predicting drug metabolizing ability according to (5), wherein the drug metabolizing ability is determined to be low when the genotype of the rs1057910 is homo or heterozygous of the mutant allele CYP2C9 * 3
(7) As the CYP2C9 * 3 detection probe, an oligonucleotide corresponding to a wild-type allele in the polymorphism specified by rs1057910 and an oligonucleotide corresponding to a mutant-type allele are used (5) A data acquisition method for predicting the described drug metabolic ability.
(8) The oligonucleotide corresponding to the wild-type allele in the polymorphism specified by rs1057910 and the oligonucleotide corresponding to the mutant allele have the same length or a difference of 2 bases or less. A data acquisition method for predicting drug metabolizing ability as described in (5).
(9) The data acquisition method for predicting drug metabolism ability according to (5), wherein the microarray according to (4) is used.
(10) The nucleic acid fragment obtained in the step of amplifying the nucleic acid fragment has a fluorescent label, and in the step of detecting hybridization, the fluorescence intensity value of the fluorescent label is measured (5) Data acquisition method for predicting drug metabolizing ability.
(11) The data acquisition method for predicting drug metabolism ability according to (5), wherein the drug is warfarin.

  The CYP2C9 * 3 detection probe according to the present invention can specifically hybridize to a nucleic acid fragment obtained by amplifying a region containing rs1057910 according to the genotype of rs1057910. Therefore, by using the CYP2C9 * 3 detection probe according to the present invention, the genotype of rs1057910 can be determined with high accuracy by a simple process such as hybridization.

  Further, since the DNA chip according to the present invention includes the CYP2C9 * 3 detection probe, it can be used when determining the genotype of rs1057910. That is, by using the DNA chip according to the present invention, the genotype of rs1057910 can be determined with high accuracy by simple processing.

  Furthermore, the data acquisition method for predicting drug metabolizing ability according to the present invention uses the above-described CYP2C9 * 3 detection probe, and uses the result of simple and highly accurate determination of the genotype of rs1057910 in the genome of the subject. doing. Therefore, according to the data acquisition method for predicting drug metabolism ability according to the present invention, the data can be obtained by a simple process.

  The CYP2C9 * 3 detection probe according to the present invention has a sequence corresponding to a predetermined region containing a single nucleotide polymorphism specified by rs1057910: 5′-GAGATACMTTGACCT-3 ′ (SEQ ID NO: 1, M is A or C, hereinafter Or an oligonucleotide consisting of a sequence complementary to the sequence, or consisting of a sequence having 1 to 6 bases added to both ends of the sequence or the complementary sequence, the entire length being 15 to 27 bases. It is a waste.

  In other words, the CYP2C9 * 3 detection probe according to the present invention may be an oligonucleotide having the sequence: GAGATACMTTGACCT, or 1-6 bases are added to the 3 ′ end and / or the 5 ′ end of the sequence: GAGATACMTTGACCT. It may be an oligonucleotide having the sequence described above. Here, the 1 to 6 bases that may be added to the 5 ′ end are specifically A, 5′-CA-3 ′, 5′-CCA-3 ′, 5′-TCCA-3 ′, 5 '-GTCCA-3' and 5'-GGTCCA-3 '. The bases 1 to 6 which may be added to the 3 ′ end are specifically T, 5′-TC-3 ′, 5′-TCT-3 ′, 5′-TCTC-3 ′ and 5 ′. -TCTCC-3 'and 5'-TCTCCC-3'.

  Alternatively, the CYP2C9 * 3 detection probe according to the present invention may be an oligonucleotide consisting of a complementary sequence of the sequence: GAGATACMTTGACCT, or at the 3 ′ end and / or the 5 ′ end of the complementary sequence of the sequence: GAGATACMTTGACCT. It may be an oligonucleotide having a sequence to which 1 to 6 bases are added. Here, the 1 to 6 bases that may be added to the 5 ′ end are specifically A, 5′-CA-3 ′, 5′-CCA-3 ′, 5′-TCCA-3 ′, 5 It is the complementary strand of '-GTCCA-3' and 5'-GGTCCA-3 '. The bases 1 to 6 which may be added to the 3 ′ end are specifically T, 5′-TC-3 ′, 5′-TCT-3 ′, 5′-TCTC-3 ′ and 5 ′. -Complementary strands of -TCTCC-3 'and 5'-TCTCCC-3'.

More specifically, the CYP2C9 * 3 detection probe according to the present invention is preferably an oligonucleotide having any one of the nucleotide sequences of SEQ ID NOs: 1 to 7.
5'-GAGATACMTTGACCT-3 '(SEQ ID NO: 1)
5'-AGAGATACMTTGACCTT-3 '(SEQ ID NO: 2)
5'-CAGAGATACMTTGACCTTC-3 '(SEQ ID NO: 3)
5'-CCAGAGATACMTTGACCTTCT-3 '(SEQ ID NO: 4)
5'-TCCAGAGATACMTTGACCTTCTC-3 '(SEQ ID NO: 5)
5'-GTCCAGAGATACMTTGACCTTCTCC-3 '(SEQ ID NO: 6)
5'-GGTCCAGAGATACMTTGACCTTCTCCC-3 '(SEQ ID NO: 7)

  rs1057910 is a single nucleotide polymorphism located within the CYP2C9 gene, the wild type is A, the wild type allele: CYP2C9 * 1, the mutant type is C, and the mutant allele: CYP2C9 * 3 . Mutant allele: CYP2C9 * 3 is known as a mutation that reduces the metabolic capacity of drugs. Therefore, it is known that an individual having a mutant allele CYP2C9 * 3 homo or hetero is delayed in drug metabolism as compared to an individual having a wild type allele.

  Here, examples of drugs in which CYP2C9 is involved in metabolism include, for example, warfarin (trade name: warfarin), ibuprofen, diclofenac, phenytoin, tolbutamide, losartan, sulfamethoxazole, torasemide, glibenclamide, glipizide, celecoxib, glimepiride, Δ9 -Tetrahydrocannabinol, fluvastatin, fluoxetine, hexobarbital, non-steroidal anti-inflammatory analgesics (piroxicam, flurbiprofen, mefenamic acid, celecoxip) and the like.

  The CYP2C9 * 3 detection probe according to the present invention is preferably a nucleic acid, more preferably DNA. The DNA includes both double strands and single strands, but the CYP2C9 * 3 detection probe according to the present invention is preferably single-stranded DNA.

  The CYP2C9 * 3 detection probe according to the present invention can be obtained by chemically synthesizing with a nucleic acid synthesizer, for example. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer or the like can be used.

  The CYP2C9 * 3 detection probe according to the present invention is preferably used in the form of a microarray (for example, a DNA chip) by immobilizing its 5 'end on a carrier. At this time, the microarray is for detecting a pair of CYP2C9 * 3 consisting of an oligonucleotide having a sequence corresponding to adenine, which is a wild-type allele of rs1057910, and an oligonucleotide having a sequence corresponding to cytosine, which is a mutant allele. It is preferable to have a probe. By using a pair of CYP2C9 * 3 detection probes corresponding to wild type and mutant type, rs1057910 is adenine homozygous, cytosine homozygous, adenine and cytosine heterozygous It can be accurately identified.

  Here, among the genotypes of rs1057910, an oligonucleotide having a sequence corresponding to adenine which is a wild type allele and an oligonucleotide having a sequence corresponding to cytosine which is a mutant allele are within 2 bases in length. Preferably, the lengths are the same.

  In addition, the microarray may have only an oligonucleotide having a sequence corresponding to cytosine, which is a mutant type among the genotypes in rs1057910, as a CYP2C9 * 3 detection probe. When at least one allele of rs1057910 is cytosine, that is, when rs1057910 genotype is C / C (cytosine homozygous) or A / C (adenine and cytosine heterozygous), drug metabolism is slow This is because it can be judged.

  As the material for the carrier, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as graphite and carbon typified by carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials represented by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, Inorganic materials such as stellite and photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol , Polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide And organic materials such as polysulfone. The shape of the carrier is not particularly limited, but is preferably a flat plate shape.

  In the present invention, a carrier having a carbon layer and a chemical modification group on the surface is preferably used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of the substrate made of the carbon layer. As the material for the substrate, those known in the art can be used, and there is no particular limitation, and the same materials as those mentioned above as the carrier material can be used.

  In the microarray according to the present invention, a carrier having a fine flat plate structure is preferably used. The shape is not limited to a rectangle, a square, or a round shape, but a shape of 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is usually used. Since it is easy to produce a carrier having a fine flat plate structure, it is preferable to use a substrate made of a silicon material or a resin material. In particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon has a slightly different orientation of the crystal axis in some parts (sometimes called a mosaic crystal), or includes atomic scale disturbances (lattice defects) Are also included.

  In the present invention, the carbon layer formed on the substrate is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene) , Carbon nanotubes), a mixture thereof, or a laminate of them is preferably used. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, the soft diamond is a generic term for an incomplete diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The carbon layer has excellent chemical stability, can withstand subsequent reactions in the introduction of chemical modification groups and binding to the analyte, and the binding is flexible because of the electrostatic binding to the analyte. It is advantageous in that it has the property of being transparent, it is transparent to the detection system UV because there is no UV absorption, and it can be energized during electroblotting. Further, it is advantageous in that nonspecific adsorption is small in the binding reaction with the analyte. As described above, a carrier whose substrate itself is made of a carbon layer may be used.

  In the present invention, the carbon layer can be formed by a known method. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionization deposition method, arc Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

  In the high-frequency plasma CVD method, a raw material gas (methane) is decomposed by glow discharge generated between electrodes by high frequency, and a carbon layer is synthesized on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The carbon layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of the remaining methane gas.

  In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

  In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

  When the carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually a monomolecular layer to about 100 μm. If it is too thin, the surface of the base substrate may be locally exposed, and conversely thick. In this case, the productivity is deteriorated, so that the thickness is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

  By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the oligonucleotide probe can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.

  An amino group can be introduced, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiation with ultraviolet rays in chlorine gas, and further irradiated with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

  The introduction of the carboxyl group can be carried out, for example, by reacting an appropriate compound with the carbon layer aminated as described above. As a compound used for introducing a carboxyl group, for example, represented by the formula: X-R1-COOH (wherein X represents a halogen atom and R1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms) Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R2-COOH (formula R2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid Polycarboxylic acid such as polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; Formula: R3-CO-R4-COOH (wherein R3 is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms) , R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms) A dicarboxylic acid represented by the formula: X-OC-R5-COOH (wherein X represents a halogen atom, R5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms) Monohalides, such as succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

  Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

  Introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

  Introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

  The active ester group means an ester group having an electron-withdrawing group having high acidity on the alcohol side of the ester group and activating the nucleophilic reaction, that is, an ester group having high reaction activity. The ester group has an electron-withdrawing group on the alcohol side of the ester group and is activated more than the alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as. More specifically, examples of the active ester group include a p-nitrophenyl group, an N-hydroxysuccinimide group, a succinimide group, a phthalimide group, and a 5-norbornene-2,3-dicarboximide group. In particular, an N-hydroxysuccinimide group is preferably used.

  The introduction of the active ester group is performed, for example, by converting the carboxyl group introduced as described above into a dehydrating condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N- It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

  The polymorphism detection probe according to the present invention is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is supported by a spotter device or the like. By spotting above, a microarray in which the polymorphism detection probe is immobilized on a carrier can be produced. Alternatively, the spotting solution may be spotted manually with a micropipette.

  Incubation is preferably performed after the spotting to allow the polymorphism detection probe to proceed with the reaction to bind to the carrier. Incubation is usually performed at a temperature of −20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed under a high humidity atmosphere, for example, under conditions of a humidity of 50 to 90%. Following the incubation, it is preferable to perform washing using a washing solution (for example, 50 mM TBS / 0.05% Tween20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) to remove DNA not bound to the carrier. .

  In addition, by using the CYP2C9 * 3 detection probe or microarray, the genotype of the single nucleotide polymorphism identified by rs1057910 in the subject (drug administration subject) can be determined. In the present invention, the step of extracting DNA from a sample derived from a subject, the step of amplifying a region containing rs1057910 using the extracted DNA as a template, and the above-mentioned CYP2C9 * 3 detection probe or microarray are used for amplification. Identifying a genotype of a single nucleotide polymorphism specified by rs1057910 contained in the nucleic acid.

  The subject is usually a human and is not particularly limited to the race or the like, but is particularly a yellow race, preferably an East Asian race, particularly preferably a Japanese. The sample derived from the subject is not particularly limited. For example, blood-related samples (blood, serum, plasma, etc.), lymph fluid, feces, cancer cells, tissue or organ crushed materials and extracts can be mentioned.

  First, DNA is extracted from a sample collected from a subject. The extraction means is not particularly limited. For example, a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB or the like can be used.

  Next, an amplification reaction is performed using the obtained DNA as a template to amplify a region containing rs1057910, preferably DNA. As the amplification reaction, polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids) method or the like can be applied. In the amplification reaction, it is desirable to add a label so that the amplified region can be identified. At this time, the method for labeling the amplified nucleic acid is not particularly limited. For example, a method in which a primer used in the amplification reaction is labeled in advance may be used, or a labeled nucleotide is used as a substrate in the amplification reaction. You may use the method to do. The labeling substance is not particularly limited, and radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

  This reaction system consists of a buffer necessary for nucleic acid amplification and labeling, a heat-resistant DNA polymerase, a primer specific to the amplification region, and a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate added with a fluorescent label, etc.) , A reaction system containing nucleotide triphosphate and magnesium chloride.

The primer used in the amplification reaction is not particularly limited as long as it can specifically amplify the region containing rs1057910, and can be appropriately designed by those skilled in the art. For example,
Primer 1: 5'-GAACGTGTGATTGGCAGAAA-3 '(SEQ ID NO: 8) and Primer 2: 5'-TCGAAAACATGGAGTTGCAG-3' (SEQ ID NO: 9)
A set of primers consisting of

  The nucleic acid fragment amplified by the primer is not particularly limited as long as it contains rs1057910. For example, it is preferably 1 kbp or less, more preferably 800 bp or less, still more preferably 400 bp or less, particularly preferably 200 bp or less.

  A hybridization reaction between the amplified nucleic acid obtained as described above and the CYP2C9 * 3 detection probe according to the present invention is performed, and the amount of nucleic acid hybridized to the CYP2C9 * 3 detection probe is detected, for example, a label. Can be measured. For example, when a fluorescent label is used, the signal intensity from the label can be quantified by detecting the fluorescent signal using a fluorescent scanner and analyzing the detected signal with image analysis software. The amplified nucleic acid hybridized with the CYP2C9 * 3 detection probe can also be quantified, for example, by preparing a calibration curve using a sample containing a known amount of DNA. The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed.For example, after hybridization at 50 ° C. for 16 hours, 2 × SSC / 0.2% SDS, 25 This refers to the conditions for washing at 5 ° C for 10 minutes and 2 x SSC at 25 ° C. Alternatively, the hybridization temperature can be 45 to 60 ° C. when the salt concentration is 0.5 × SSC. If the chain length of the CYP2C9 * 3 detection probe is short, the hybridization temperature is lower. More preferably, when the chain length of the CYP2C9 * 3 detection probe is long, the hybridization temperature is more preferably higher. It goes without saying that the hybridization temperature having specificity increases as the salt concentration increases, whereas the hybridization temperature having specificity decreases as the salt concentration decreases.

  In the above hybridization reaction, a method is preferred in which a microarray in which the CYP2C9 * 3 detection probe according to the present invention is immobilized on a carrier is used and a solution containing an amplified nucleic acid is applied to the microarray.

  Based on the amount of the amplified nucleic acid hybridized to the CYP2C9 * 3 detection probe according to the present invention, the data acquisition method for predicting drug metabolic ability according to the present invention has the genotype of rs1057910 in the subject's genome, Specify whether the mutant allele is homozygous or heterozygous. Then, when the genotype of rs1057910 has a mutant allele: CYP2C9 * 3 in homo or hetero, it can be determined that the subject has a slow drug metabolism rate. For this reason, the individual having the mutant allele CYP2C9 * 3 as a homozygote and the individual having a heterozygote as a heterozygote can reduce the dose of the above-mentioned drug compared to an individual having the wild-type allele as a homozygote.

  In particular, when comparing individuals with mutant allele: CYP2C9 * 3 homozygous and individuals with mutant allele: CYP2C9 * 3 heterozygous, drugs in individuals with mutant allele: CYP2C9 * 3 homozygous It can be judged that the metabolic rate is slower. Therefore, the drug dose for an individual who has a mutant allele: CYP2C9 * 3 in a homozygous state can be made smaller than the drug dose for an individual who has a mutant allele: CYP2C9 * 3 hetero.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, a DNA chip having a pair of CYP2C9 * 3 detection probes for discriminating the genotype of rs1057910 was produced. A list of nucleic acid probes mounted on the DNA chip is shown in Table 1.

  In this example, a nucleic acid fragment containing rs1057910 having a fluorescent label is amplified by PCR, and the resulting nucleic acid fragment is allowed to act on the DNA chip prepared as described above to detect nucleic acid fragments and polymorphisms. Hybridization with the probe was detected by fluorescence intensity.

  The fluorescently labeled primer was prepared by using an oligonucleotide C2C93-R (Life Technologies Japan) shown in Table 2 labeled with an amino group at the 5 ′ end and IC5-OSu (manufactured by Dojindo Laboratories) in phosphate buffer (pH 8). .0), the mixture was reacted at 4 ° C. for 1 day and then purified using MicroSpin G-25 Columns (manufactured by GE Healthcare). By measuring the absorbance at 260 nm and 640 nm of the purified product, the concentration of the primer and the concentration of the fluorescent dye were calculated.

  A fluorescent unlabeled primer (C2C93-F) was added to the obtained fluorescently labeled primer, and primers of the concentrations shown in Table 2 were mixed in equal amounts to prepare a primer mix. PCR was performed under the conditions shown in Table 3. Specifically, PCR was prepared by preparing a reaction solution with the composition shown in Table 4 and preparing a plasmid DNA having a wild type sequence, a plasmid DNA having a mutant type sequence, and a hetero equivalent prepared as 2 pg / μL as a wild type specimen. The plasmid DNA was added to each reaction tube in an amount of 5 μL, and PCR was performed using a thermal cycler (manufactured by ABI).

  When hybridizing the amplification product obtained as described above to a DNA chip, first, a wet box is placed in a chamber set at a specified temperature (any one of 45, 50, 55, and 60 ° C.). The chamber and the box were pre-heated sufficiently. In a new sample tube, 1.5 μL of hybridization buffer (2.25 × SSC / 0.23% SDS) and 3 μL of amplification product were added and mixed. 3 μL of this mixture was dropped onto the DNA chip, covered, placed in a fully preheated wet box, and the wet box was placed in the chamber. Thereafter, the DNA chip and the amplification product were reacted at a specified temperature for 1 hour.

  Thereafter, the cover was immediately removed, and the plate was left in a 1 × SSC / 0.1% SDS solution for 5 minutes, and then left in a 1 × SSC solution at room temperature until detection. BIOSHOT (manufactured by Toyo Kohan Co., Ltd.) was used for detection of hybridization. At this time, the exposure time was set to 5 seconds. Further, the median value was obtained from the numerical values of the pixels within each spot diameter. For the same probe spot, the average of the median values was obtained and used as the output value of each spot.

  In the evaluation of specificity, a determination value obtained by substituting the fluorescence intensity value of each spot into the following determination formula was used as an index.

  Table 5 shows the determination values obtained by changing the hybridization temperature in this example. Table 6 shows the determination values obtained when a pair of CYP2C9 * 3 detection probes corresponding to the wild-type allele and the mutant-type allele have different base lengths.

  As can be seen from Table 5, with the 15-27 base CYP2C9 * 3 detection probe designed in this example, the determination value obtained from each genotype is 0.2 or more at a hybridization reaction temperature of 45-60 ° C. It was revealed that genotyping was possible. Moreover, as shown in Table 6, the CYP2C9 * 3 detection probe of 15 to 27 bases designed in this example has specificity even when the base length is different by 2 bases. confirmed.

Claims (11)

  Sequence: 5′-GAGATACMTTGACCT-3 ′ (SEQ ID NO: 1, M is A or C) or a sequence complementary to the sequence, or 1 to 6 bases added to both ends of the sequence or the complementary sequence A probe for CYP2C9 * 3 detection, comprising an oligonucleotide consisting of a sequence of 15 to 27 bases in length.   The base sequence of the oligonucleotide is A, 5'-CA-3 ', 5'-CCA-3', 5'-TCCA-3 ', 5'-GTCCA at the 5' end of the base sequence shown in SEQ ID NO: 1. -3 ′ and 5′-GGTCCA-3 ′ are added, and T, 5′-TC-3 ′, 5′-TCT-3 ′, 5′-TCTC-3 ′ are added to the 3 ′ end of the above sequence. 2. The CYP2C9 * 3 according to claim 1, which is a base sequence to which any of 5′-TCTCC-3 ′ and 5′-TCTCCC-3 ′ is added or a complementary sequence to the base sequence. Probe for detection. The CYP2C9 * 3 detection probe according to claim 1, wherein the base sequence of the oligonucleotide is any one of SEQ ID NOs: 2 to 7, or a complementary sequence to the base sequence.
AGAGATACMTTGACCTT (SEQ ID NO: 2)
CAGAGATACMTTGACCTTC (SEQ ID NO: 3)
CCAGAGATACMTTGACCTTCT (SEQ ID NO: 4)
TCCAGAGATACMTTGACCTTCTC (SEQ ID NO: 5)
GTCCAGAGATACMTTGACCTTCTCC (SEQ ID NO: 6)
GGTCCAGAGATACMTTGACCTTCTCCC (SEQ ID NO: 7)   A microarray comprising the CYP2C9 * 3 detection probe according to any one of claims 1 to 3. Amplifying a nucleic acid fragment containing a polymorphic site specified by rs1057910 in the CYP2C9 gene using a subject-derived genome as a template;
Contacting the obtained nucleic acid fragment with the CYP2C9 * 3 detection probe according to any one of claims 1 to 3,
A step of detecting hybridization between the obtained nucleic acid fragment and the CYP2C9 * 3 detection probe,
A data acquisition method for predicting drug metabolizing ability to determine the genotype of rs1057910.   6. The method for obtaining data for predicting drug metabolizing ability according to claim 5, wherein the drug metabolizing ability is determined to be low when the genotype of rs1057910 is homozygous or heterozygous for the mutant allele CYP2C9 * 3.   6. The drug according to claim 5, wherein an oligonucleotide corresponding to the wild type allele in the polymorphism specified by rs1057910 and an oligonucleotide corresponding to the mutant allele are used as the CYP2C9 * 3 detection probe. Data acquisition method for predicting metabolic capacity.   The oligonucleotide corresponding to the wild-type allele in the polymorphism specified by rs1057910 and the oligonucleotide corresponding to the mutant-type allele have the same length or a difference within 2 bases 6. A data acquisition method for predicting drug metabolic capacity according to claim 5.   6. The data acquisition method for predicting drug metabolic capacity according to claim 5, wherein the microarray according to claim 4 is used.   6. The drug metabolism according to claim 5, wherein the nucleic acid fragment obtained in the step of amplifying the nucleic acid fragment has a fluorescent label, and the fluorescence intensity value of the fluorescent label is measured in the step of detecting hybridization. Data acquisition method for predicting performance.   6. The data acquisition method for predicting drug metabolizing ability according to claim 5, wherein the drug is warfarin.