CN101760528B - Medicine metabolic relevant loci detection method - Google Patents
Medicine metabolic relevant loci detection method Download PDFInfo
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- CN101760528B CN101760528B CN200810208219A CN200810208219A CN101760528B CN 101760528 B CN101760528 B CN 101760528B CN 200810208219 A CN200810208219 A CN 200810208219A CN 200810208219 A CN200810208219 A CN 200810208219A CN 101760528 B CN101760528 B CN 101760528B
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Abstract
The invention relates to a medicine metabolic relevant loci detection method, which comprises the following steps: extracting genome DNA from human samples; respectively designing a Taqman probe pair and a primer pair according to at least two medicine metabolic relevant genes; respectively marking the 5' end and the 3'end of the Taqman probe pair with fluorescence reporting genes and fluorescence quenching genes; carrying out fluorescence quantitative PCR augmentation on the genome DNA; and judging whether the medicine metabolic relevant genes have the mutation according to the fluorescence quantitative PCR augmentation results. Preferably, the number of the medicine metabolic relevant genes is four, the Taqman probe pair and the primer pair are used for detecting a loci rs1057910 of a gene CYP2C9, a loci rs4244285 of a gene CYP2C19, a loci rs4986893of a gene CYP2C19, a loci rs1065852 of a gene CYP2D6 and a loci rs28371759 of a gene CYP3A4. The invention has the advantages of ingenious design, simple operation and accurate and reliable detection results, and provides the reference frame for determining whether professional doctors are needed to be consulted so as to make sure the medicine can be taken or not or the proper dosage and the like when a certain medicine is taken.
Description
Technical field
The present invention relates to function related locus detection technique field, more specifically, relate to medicament metabolism ability related locus detecting method technical field, be meant a kind of drug metabolism related locus detecting method especially.
Background technology
(cytochrome P450 CYP), claims mixed-function oxidase (mixed functionoxidase) and monooxygenase (monooxygenase) again to cytochrome P 450 enzymes; Mainly be present in the hepatomicrosome; In the exogenous compounds bio-transformation of (comprising medicine and poisonous substance), play crucial effect, the metabolic rate of its activity decision medicine has direct relation with the clearance rate of medicine; Be the phasel enzyme of drug metabolism, thereby be called drug metabolism enzyme again.Many kinds of medicines of its energy metabolism and/or activation, and procarcinogen, preceding poisonous substance and mutagen.The gene of P450 enzyme system mainly contains CYP1, CYP2, and three families of CYP3, relevant have following several kinds of important P450 enzymes: CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4.
Human many drug metabolism CYP450 enzymes have heritable variation, and research shows, many hypotypes of CYP450 enzyme as: the activity of enzymes such as CYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2D6 and CYP2E1 distributes the crowd and is genetic polymorphism.The polymorphum of cytochrome P 450 enzymes is to cause individual important factor to medicament metabolism ability difference; We are divided into the liver metabolism ability according to person under inspection's genotype: not good figure metabolizers (PM), osculant metabolizers (IM), extensively four types of type metabolizers (EM) and super type metabolizers (UM); The liver metabolism ability more a little less than; Various kinds of drug is big more to hepatotoxicity, and the power of liver drug metabolic capacity is determined by each subtype gene type of cytochrome P 450 enzymes.The corresponding different drug of the different loci of cytochrome P 450 enzymes; Comprise anti-inflammation analgesia medicine (diclofenac, for sieve former times health, Ibuprofen BP/EP, Naproxen Base, piroxicam, mefenamic acid, FLURBIPROFEN USP24, indomethacin), antiepileptic drug (Phenytoin Sodium Salt, Carbamzepine), anticoagulant (warfarin, temparin), hypoglycemic agents (tolbutamide, Glipizide, Glyburide, glimepiride), other (losartan, torasemide, tamoxifen, dronabinol, amitriptyline) etc. like the associated metabolic medicine of CYP2C9 gene; And the enzyme clearance rate of CYP2C9 variation has only 15%～20% of wild-type; Be prone to produce the substrate Side effects of pharmaceutical drugs; Especially warfarin, Phenytoin Sodium Salt and some antidiabetic drug medicines (like Glyburide, glimepiride); Cause bleeding like the S-warfarin, tolbutamide causes hypoglycemic shock.Therefore, as run under the situation of not good figure metabolizers (PM) and osculant metabolizers (IM), medication please consulting profession doctor suggestion.
Therefore; In order earlier to understand medicament metabolism ability, and then when taking some drugs, accomplish to shoot the arrow at the target, avoid some drugs to cause spinoff; Need to go out through the accurate examination of gene test in advance the situation of said medicine metabolism related locus; Thereby understand individual gene physique and liver metabolism ability metabolism intensity, assist the possible drug reaction of prediction, via the judgement of medical practitioner to medicine; The most suitable your list of medications is provided, and the untoward reaction of avoiding maybe unconformable medicine being caused takes place.
Summary of the invention
Main purpose of the present invention is exactly the problems and shortcomings to above existence; A kind of drug metabolism related locus detecting method is provided; The design of this detection method is ingenious, simple to operate, detected result accurately and reliably, whether need when taking some drugs the consulting profession doctor reference frame to be provided with determine whether to take and taking dose etc.
To achieve these goals, the technical scheme of the present invention's employing is following:
This drug metabolism related locus detecting method is characterized in, comprises step:
A. extract genomic dna from people's sample;
B. design a pair of Taqman probe respectively to right according at least 2 drug metabolism genes involveds with a pair of primer; 5 ' end that said Taqman probe is right and 3 ' end be mark fluorescent reporter group and fluorescent quenching group respectively, respectively said genomic dna is carried out fluorescent quantitative PCR;
C. judge according to the fluorescent quantitative PCR result whether said drug metabolism genes involved undergos mutation.
Preferably, the number of said drug metabolism genes involved is 4.
More preferably, said drug metabolism genes involved is CYP2C9 gene, CYP2C19 gene, CYP2D6 gene and CYP3A4 gene.
Further; Said Taqman probe to said primer to being used to detect the rs1057910 site of CYP2C9 gene; The rs4244285 site of CYP2C19 gene; The rs4986893 site of CYP2C19 gene, the rs1065852 site of CYP2D6 gene, the rs28371759 site of CYP3A4 gene.
Preferably; Said Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:1 and the SEQ ID NO:2; 5 ' end difference flag F AM mark and the HEX mark that said Taqman probe is right; The equal mark TAMRA mark of 3 ' end, said primer is to being the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.
Preferably; Said Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:5 and the SEQ ID NO:6; 5 ' end difference flag F AM mark and the HEX mark that said Taqman probe is right; The equal mark TAMRA mark of 3 ' end, said primer is to being the nucleotide sequence shown in SEQ ID NO:7 and the SEQ ID NO:8.
Preferably; Said Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:9 and the SEQ ID NO:10; 5 ' end difference flag F AM mark and the HEX mark that said Taqman probe is right; The equal mark TAMRA mark of 3 ' end, said primer is to being the nucleotide sequence shown in SEQ ID NO:11 and the SEQ ID NO:12.
Preferably; Said Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:13 and the SEQ ID NO:14; 5 ' end difference flag F AM mark and the HEX mark that said Taqman probe is right; The equal mark TAMRA mark of 3 ' end, said primer is to being the nucleotide sequence shown in SEQ ID NO:15 and the SEQ ID NO:16.
Preferably; Said Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:17 and the SEQ ID NO:18; 5 ' end difference flag F AM mark and the TET mark that said Taqman probe is right; The equal mark TAMRA mark of 3 ' end, said primer is to being the nucleotide sequence shown in SEQ ID NO:19 and the SEQ ID NO:20.
Innovative point of the present invention is that the relevant marquis of several drug metabolisms examines the integration in site; Marquis through choosing a plurality of drug metabolisms examines the site; According to CYP2C9 gene, CYP2C19 gene, CYP2D6 gene and CYP3A4 gene design Auele Specific Primer and Taqman probe the genomic dna from people's sample is carried out fluorescent quantitative PCR, can know that according to the amount of the fluorescence that produces and the kind of fluorescence the marquis examines the genotype in site, design ingenious, simple to operate, detected result accurately and reliably; Thereby understand individual gene physique and liver metabolism ability metabolism intensity as early as possible to medicine; Assist the possible drug reaction of prediction, thereby when needs are taken some drugs, running under the situation of not good figure metabolizers (PM) and osculant metabolizers (IM); The suggestion of medical consultation medical practitioner; Via the judgement of medical practitioner, optimal list of medications is provided, the untoward reaction of avoiding the unconformable medicine of possibility to be caused takes place.Generally speaking, be the osculant of certain medicine or good figure metabolizers not if the gene test result shows the person under inspection, this person under inspection answers decrement to take when using this medicine; If the person under inspection is extensive type metabolizers, this person under inspection please takes by doctor's advice when using this medicine; If the person under inspection is super type metabolizers, when using this medicine, this person under inspection please use by dosage.
Embodiment
For better understanding content of the present invention, be described further below in conjunction with specific embodiment.
1. extracting genome DNA:
1.1 reagent and instrument:
1.1.1 reagent and consumptive material: genome DNA extraction test kit (TIANamp BLOOD DNA kit, DP318-03; TIANamp Micro micro-example genome DNA extracting reagent kit, DP316; TIANamp buccal swab genome DNA extracting reagent kit DP322-03) contains: cell pyrolysis liquid CL; Damping fluid GA; Damping fluid GS; Damping fluid GB; Damping fluid GD; Rinsing liquid PW; Elution buffer TB; Carrier RNA; RNase-free ddH2O; Proteinase K (20mg/ml); Adsorption column; Collection tube (2ML); 1.5ML aseptic collection tube; Absolute ethyl alcohol.
1.1.2 instrument: DENVILLE260 whizzer, XW-80A vortex mixer, H.H.S pointer-type electric-heated thermostatic water bath.
1.2 extraction step
From whole blood, extract genomic dna
1.2.1 the anticoagulated whole blood of getting 200 μ l if the amount of sample is less than 200 μ l, then adds damping fluid GS and is supplemented to 200 μ l to the Eppendorf pipe of 1.5ml.If sample size more than 200 μ l, needs to handle with cell pyrolysis liquid CL, concrete steps are following: the cell pyrolysis liquid CL that in sample, adds 1-2.5 times of volume; Put upside down mixing, 10, centrifugal 1 minute of 000rpm; Draw supernatant, stay nucleus deposition (, can repeat above step once) if cracking is not thorough; In the centrifugal nucleus deposition of collecting, add 200 μ l damping fluid GS, vibration is to thorough mixing.
1.2.2 add the Proteinase K solution of 20 μ l, mixing adds the damping fluid GB of 200 μ l again, fully puts upside down mixing at once.56 ℃ of water-bath samples 10 minutes please whenever softly be put upside down the mixing sample up and down at a distance from 3 minutes in the water-bath process, and the solution strain is limpid.(thoroughly do not become limpid like solution, can prolong cracking time to solution becomes limpid till).
1.2.3 add 200 μ l absolute ethyl alcohols, fully put upside down mixing, at this moment flocks may appear.
All add in the adsorption column (adsorption column is put into collection tube) 1.2.4 will go up step gained solution and a flocks, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, adsorption column is put into collection tube.
1.2.5 in adsorption column, add 500 μ l damping fluid GD (please checking to have added absolute ethyl alcohol whether earlier before using), 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, adsorption column is put into collection tube.
1.2.6 in adsorption column, add 700 μ l rinsing liquid PW (please checking to have added absolute ethyl alcohol whether earlier before using), 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, adsorption column is put into collection tube.
1.2.7 in adsorption column, add 500 μ l rinsing liquid PW, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube.
1.2.8 adsorption column is put back in the collection tube, 12,000rpm (～13,400 * g) centrifugal 2 minutes, outwell waste liquid.Adsorption column places room temperature to place several minutes, thoroughly to dry rinsing liquid remaining in the sorbing material.
1.2.9 adsorption column is changed in the clean centrifuge tube, and to adsorption film mid-way unsettled Dropwise 5 0-200 μ l elution buffer TB, room temperature was placed 2-5 minute, and 12,000rpm (～13,400 * g) centrifugal 2 minutes, solution is collected in the centrifuge tube.
Attention: the elution buffer volume should not be less than 50 μ l, the too small organic efficiency that influences of volume.For increasing the yield of genomic dna, can the centrifugal solution that obtains be added in the adsorption column again room temperature placement 2 minutes, 12,000rpm (～13,400 * g) centrifugal 2 minutes.The pH of elutriant has very big influence for elution efficiency.
Should guarantee that its pH value (can be transferred to this scope with the pH value of water with NaOH) in the 7.0-8.5 scope if water is cooked elutriant, the pH value is lower than 7.0 and understands the reduction elution efficiencies; And the DNA product should be kept at-20 ℃, in case dna degradation.
2.Taqman probe method detects the marquis and examines the site:
2.1 experiment reagent and instrument
2.1.1 experiment reagent
Fluorescent quantitation reagent: ABI TaqMan 2 * PCR Master mix (P/N:4326614, Lot:G15502).
Regular-PCR reagent: the Ex Taq archaeal dna polymerase of precious biotechnology (Dalian) ltd (P/N:DRR100B, Lot:CKA1801A).
2.1.2 laboratory apparatus: ABI 9700 type PCR appearance and ABI 7900HT type quantitative real time PCR Instrument.
2.1.3 detection site probe and primer table
2.2 experimental procedure
2.2.1 the concentration of all sample DNAs is adjusted to 20ng/ul～50ng/ul.
2.2.2 the site is detected
The a.PCR reaction system:
The b.PCR cycling condition:
2.2.3 judge the genotype of corresponding candidate locus according to the kind of the amount of the fluorescence that discharges in the reaction process and fluorescence.
3. interpretation of result:
Analyze according to the final terminal point fruit somatotype figure that reads to harden, the result is following:
Reaction 1: be used to detect the rs1057910 site of CYP2C9 gene, the associated metabolic medicine comprises: anti-inflammation analgesia medicine (diclofenac, for sieve former times health, Ibuprofen BP/EP, Naproxen Base, piroxicam, mefenamic acid, FLURBIPROFEN USP24, indomethacin), antiepileptic drug (Phenytoin Sodium Salt, Carbamzepine), anticoagulant (warfarin, temparin), hypoglycemic agents (tolbutamide, Glipizide, Glyburide, glimepiride), other (losartan, torasemide, tamoxifen, dronabinol, amitriptyline etc.).There are three kinds of genotype: AA in this time inspection site in the crowd; AC and CC; Wherein carrying the AA genotype is that normal population is that extensive type metabolizers is represented with EM; The liver metabolism ability that the crowd who carries AC carries the genotypic crowd of AA is wanted weak and is represented with IM, and the crowd who carries CC carries that preceding two kinds of genotype crowds' liver metabolism ability is the most weak representes that with PM the strong and weak size of liver metabolism ability is AA type＞AC type＞CC type; Somatotype map analysis result shows that the rs1057910 site is AA, is extensive type metabolizers EM;
Reaction 2: be used to detect the rs4244285 site of CYP2C19 gene, the associated metabolic medicine comprises: antimalarial drug (chloroguanide), thymoleptic (amitriptyline, clomipramine, imipramine, citalopram), anticonvulsive drug (PHT, Phenytoin Sodium Salt, ethotoin, diazepam, phenylethyl barbituric acid, Mephogarbital), anti-ulcerative drug (omeprazole, lansoprazole), other (Hexobarbital, Proprasylyte, carisoprodol, thioridazine, warfarin etc.).There are three kinds of genotype: GG in this time inspection site in the crowd; AG and AA; Wherein carrying the GG genotype is that normal population is that extensive type metabolizers is represented with EM; Carry liver metabolism ability that the genotypic crowd of AG carries the genotypic crowd of GG and want weak and represent with IM, the crowd who carries AA carries that preceding two kinds of genotype crowds' liver metabolism ability is the most weak representes that with PM the strong and weak size of liver metabolism ability is GG type＞AG type＞AA type; Somatotype map analysis result shows that the rs4244285 site is GA, is osculant metabolizers IM;
Reaction 3: be used to detect the rs4986893 site of CYP2C19 gene, the associated metabolic medicine comprises: antimalarial drug (chloroguanide), thymoleptic (amitriptyline, clomipramine, imipramine, citalopram), anticonvulsive drug (PHT, Phenytoin Sodium Salt, ethotoin, diazepam, phenylethyl barbituric acid, Mephogarbital), anti-ulcerative drug (omeprazole, lansoprazole), other (Hexobarbital, Proprasylyte, carisoprodol, thioridazine, warfarin etc.).There are three kinds of genotype: GG in this time inspection site in the crowd; AG and AA; Wherein carrying the GG genotype is that normal population is that extensive type metabolizers is represented with EM; Carry liver metabolism ability that the genotypic crowd of AG carries the genotypic crowd of GG and want weak and represent with IM, the crowd who carries AA carries that preceding two kinds of genotype crowds' liver metabolism ability is the most weak representes that with PM the strong and weak size of liver metabolism ability is GG type＞AG type＞AA type; Somatotype map analysis result shows that the rs4986893 site is GG, is extensive type metabolizers EM;
Reaction 4: be used to detect the rs1065852 site of CYP2D6 gene, the associated metabolic medicine comprises: anodyne (oxycodone, dihydrocodeine, morphine monomethyl ether, U-26225A), psychotroptic drug (risperidone, thioridazine, trilafon, leoponex, trifluperidol, Fluphenazine, CHLORPROMAZINE HCL, haloperidol), thymoleptic (clomipramine, nortriptyline, amitriptyline, imipramine, Trimipramine, Pertofran, zolpidem, amiflamine, fluoxetine, paroxetine, fluvoxamine, tomoxetine (treatment hyperkinetic syndrome), trazodone, Venlafaxine VEN), the normal medicine of anti-rhythm of the heart husband (Propafenone, mexiletine, flecainide, En Nika, sparteine, Ajmaline, Lilly 99170), beta-blockers (metoprolol, pindolol, timolol, carvedilol, bufuralol, Proprasylyte, bufetolol, Sonorm, alprenolol), antihypertensive drug (Debrisoquine, Wy-21901, envacar, urapidil, nicergoline), other (Dextromethorphane Hbr, Ethylmorphine, Sch 31846, tacrine, piperazine Ke Xilin, phenformin, LT, ondansetron, promethazines etc.).There are three kinds of genotype: CC in this time inspection site in the crowd; CT and TT; It is genotypic for normal population is that extensive type metabolizers is represented with EM to carry CC and CT, carries liver metabolism ability that the genotypic crowd of TT carries CC and the genotypic crowd of CT and wants weak and represent that with IM the strong and weak size of liver metabolism ability is CC type and CT type＞TT type; Somatotype map analysis result shows that the rs1065852 site is CT, is extensive type metabolizers EM;
Reaction 5: the rs28371759 site that is used to detect the CYP3A4 gene; Associated metabolic medicine: microbiotic (Oxacyclotetradecane,erythromycin deriv, clarithromycin, troleomycin, Azythromycin, Rifampin), antifungal drug (itraconazole, KETOKONAZOL), antiviral drug (Indinavir, ritonavir, Saquinavir, zidovudine), anodyne (alfentanil fentanyl, morphine monomethyl ether, Cocaine, dihydrocodeine, oxycodone, methadone, PARACETAMOL BP98, U-26225A), psychotroptic drug (haloperidol, pimozide, leoponex), thymoleptic (amitriptyline, imipramine, clomipramine, nefazodone, Sertraline, trazodone, citalopram, cyclobenzaprine), anticonvulsive drug (Carbamzepine, Trimethadione, felbamate), sedative hypnotic (triazolam, diazepam, zolpidem), the normal medicine of anti-rhythm of the heart husband (amiodarone, disopyramide, lignocaine, Quinidine, Propafenone), calcium antagonist (felodipine, nifedipine, nitrendipine, amlodipine, nicardipine, Isrodipine, verapamil, Odizem, Mibefradil), lipid regulating agent (SV, lovastatin, fluvastatin, Cerivastatin, bezafibrate, fenofibrate, gemfibrozil), antihistaminic (astemizole, terfenadine, LT, ebastine, azelastine), hormones (testosterone, androsterone, KE, gestodene, Theelin,dihydro-, DEXAMETHASONE BP98, mifepristone, methylprednisolone, Progesterone, prednisolone), antitumour drug (endoxan, ifosfamide, vinealeucoblastine(VLB), tamoxifen, vincristine(VCR), purple refined alcohol), immunosuppressor (ciclosporin, tacrolimus), other (chloroguanide, NSC-757., grapefruit juice (beverage has interaction with multiple medicine), Virga (vigour, viagra), Glyburide, R--warfarin, zileuton, ondansetron, cisapride, finasteride, flutamide, Thiopental Sodium, Sha Tuosiqiong, losartan, quinine, ropivacaine, omeprazole, dapsone, Dextromethorphane Hbr, Ethylmorphine etc.).There are three kinds of genotype: TT in this time inspection site in the crowd; CT and CC; It is genotypic for normal population is that extensive type metabolizers is represented with EM to carry TT and CT, carries liver metabolism ability that the genotypic crowd of CC carries CT and the genotypic crowd of TT and is eager to excel and representes that with UM the strong and weak size of liver metabolism ability is CC type＞TT type and CT type; Somatotype map analysis result shows that the rs28371759 site is TT, is extensive type metabolizers EM;
According to above-mentioned detected result; The rs28371759 site of rs1065852 site and CYP3A4 gene of rs4986893 site, CYP2D6 gene that can judge rs1057910 site, the CYP2C19 gene of the above-mentioned CYP2C9 gene of being examined is normal; Be extensive type metabolizers EM; Only the rs4244285 site of CYP2C19 gene is unusual; Belong to osculant metabolizers IM; The person under inspection needs suitably to note taking of antimalarial drug (chloroguanide), thymoleptic (amitriptyline, clomipramine, imipramine, citalopram), anticonvulsive drug (PHT, Phenytoin Sodium Salt, ethotoin, diazepam, phenylethyl barbituric acid, Mephogarbital), anti-ulcerative drug (omeprazole, lansoprazole), other (Hexobarbital, Proprasylyte, carisoprodol, thioridazine, warfarin) medicine of etc.ing, and consulting profession doctor during best medication is via the judgement of medical practitioner; Optimal list of medications is provided, and the untoward reaction of avoiding the unconformable medicine of possibility to be caused takes place.
In sum, drug metabolism related locus detecting method of the present invention design is ingenious, simple to operate, detected result accurately and reliably, whether need when taking some drugs the consulting profession doctor reference frame to be provided with determine whether to take and taking dose etc.
Need to prove that all documents of mentioning are in the present invention quoted as a reference in this application, just quoted such as a reference separately as each piece document.Should understand in addition; Above-described is specific embodiment of the present invention and the know-why used; After having read foregoing of the present invention; Those skilled in the art can do various changes or modification to the present invention and not deviate from spirit of the present invention and scope, and these equivalent form of values fall within the scope of the invention equally.
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<222>(1)…(25)
< 223>upstream primer that designs according to people CYP3A4 gene order
<400>19
atgccctaca?ttgatctgat?ttacc 25
<210>20
<211>20
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
<222>(1)…(20)
< 223>according to the downstream primer of people CYP3A4 sequences Design
<400>20
cgtggtttca?tagccggcaa 20
Claims (1)
1. the Taqman probe that is used for the detection of drug metabolism related locus is to right with primer; It is characterized in that, said Tagman probe to said primer to designing respectively according to 4 drug metabolism genes involveds, said drug metabolism genes involved is CYP2C9 gene, CYP2C19 gene, CYP2D6 gene and CYP3A4 gene; Said Taqman probe to said primer to being used to detect the rs1057910 site of CYP2C9 gene; The rs4244285 site of CYP2C19 gene, the rs4986893 site of CYP2C19 gene, the rs1065852 site of CYP2D6 gene; The rs28371759 site of CYP3A4 gene, wherein:
The Taqman probe in rs1057910 site that is used to detect the CYP2C9 gene is to being the nucleotide sequence shown in SEQ ID NO:1 and the SEQ ID NO:2; 5 ' end difference flag F AM mark and the HEX mark that the Taqman probe is right; The equal mark TAMRA mark of 3 ' end, primer is to being the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4;
The Taqman probe in rs4244285 site that is used to detect the CYP2C19 gene is to being the nucleotide sequence shown in SEQ ID NO:5 and the SEQ ID NO:6; 5 ' end difference flag F AM mark and the HEX mark that the Taqman probe is right; The equal mark TAMRA mark of 3 ' end, primer is to being the nucleotide sequence shown in SEQ ID NO:7 and the SEQ ID NO:8;
The Taqman probe in rs4986893 site that is used to detect the CYP2C19 gene is to being the nucleotide sequence shown in SEQ ID NO:9 and the SEQ ID NO:10; 5 ' end difference flag F AM mark and the HEX mark that the Taqman probe is right; The equal mark TAMRA mark of 3 ' end, primer is to being the nucleotide sequence shown in SEQ ID NO:11 and the SEQ ID NO:12;
The Taqman probe in rs1065852 site that is used to detect the CYP2D6 gene is to being the nucleotide sequence shown in SEQ ID NO:13 and the SEQ ID NO:14; 5 ' end difference flag F AM mark and the HEX mark that the Taqman probe is right; The equal mark TAMRA mark of 3 ' end, primer is to being the nucleotide sequence shown in SEQ ID NO:15 and the SEQ ID NO:16;
The Taqman probe in rs28371759 site that is used to detect the CYP3A4 gene is to being the nucleotide sequence shown in SEQ ID NO:17 and the SEQ ID NO:18; 5 ' end difference flag F AM mark and the TET mark that the Taqman probe is right; The equal mark TAMRA mark of 3 ' end, primer is to being the nucleotide sequence shown in SEQ ID NO:19 and the SEQ ID NO:20.
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