CN101522911A - Htsnps for determining a genotype of cytochrome P450 1a2, 2A6 and 2D6, PXR and UPD-glucuronosyltransferase 1A gene and multiplex genotyping methods using thereof - Google Patents

Abstract

The present invention relates to htSNPs for determining a genotype of cytochrome P450 1A2 (CYP1A2), 2A6 (CYP2A6) and 2D6 (CYP2D6), PXR and UDP- glucuronosyltransf erase Ia (UGTlA) genes and a gene chip using the same, and more particularly, to a selection method of htSNPs for determining a haplotype of human CYP1A2, CYP2A6, CYP2D6, PXR and UGTlA genes, a method of determining a genotype of the genes by using the htSNPs and a gene chip therefor.

Description

The method that is used for determining the haplotype tagged single-nucleotide polymorphic of Cytochrome P450 1A2,2A6 and 2D6, PXR and UDP-glucuronyl transferase 1A gene genotype and is used for multiple gene type

Technical field

Equipment of the present invention and method relate to haplotype tagged single-nucleotide polymorphic (htSNP) and the gene chip that is used for determining Cytochrome P450 1A2,2A6 and 2D6, PXR and UDP-glucuronyl transferase 1A gene genotype, more specifically, relate to be used for determining human CYP1A2, CYP2A6, CYP2D6, NR1I2 (=PXR) and the system of selection of the htSNP of the haplotype of UGT1A gene, determine the method for described gene genotype and be used for the gene chip of described method.

Background technology

For the reason of genetic diversity, individual toxicity and effect to medicine has differential responses.Therefore, consider that in the initial development stage of medicine important pharmaceutical protein is necessary with respect to the effect of genetic diversity, because this can reduce the possibility failure of drug development.Haplotype is one of factor of the genetic diversity between the decision individuality.Haplotype refers to the combination of the polymorphism of each gene order in single research population.Haplotype provides more accurate and more reliable information about genetic diversity than individual polymorphism.

Human cell's cytochrome p 450 is the part of oxyphorase, oxyphorase is assisted the external chemical substance of oxidation such as medicine, carcinogenic substance and toxin and inner material such as cholesterol, lipid acid and VITAMIN (Nelson etc., Phamacogenetics 6:1-42,1996).The various subfamilies of Cytochrome P450 in liver, kidney, small intestine and lung, have been found.

Human cell's cytochrome p 450 1A2 (hereinafter claiming CYP1A2) gene and CYP1A1 and CYP1B1 are the drug metabolism enzymes that comprises in the CYP1 gene group.CYP1A2 mainly produces in liver, and accounts for 15% of cytopigment enzyme total amount.CYP1A2 participates in the metabolism of important medical such as caffeine, leoponex (clozapine), imipramine (imiparamine) and Proprasylyte (propranolol).In addition, the inner synthetic (as 17 beta estradiols, uroporphyrinogen III) of CYP1A2 catalysis, and the bioactivation of carcinogenic substance (as the N-hydroxylation of the epoxidation and the aromatic amine/heterocyclic amine of polycyclic aromatic hydrocarbons) (Brosen K., Clinical Pharmacokinetic, 1995,29 (suppl1): 20-25; Josephy PD., Environ.Mol.Mutagen, 2001,38:12-18).Human cell's cytochrome p 450 2A6 (hereinafter claiming CYP2A6) gene is positioned on No. 19 karyomit(e), and the pseudogene CYP2A7 with closely similar gene order is on the CYP2A6 gene.The CYP2A6 enzyme is a kind of Nicotine to be converted into the main enzyme of cotinine (cotinine), and the CYP2A6 enzyme participates in about 80% nicotine metabolite.The CYP2A6 enzyme is converted into activity in vivo medicine 5 FU 5 fluorouracil (5-FU) with cancer therapy drug Tegafur (tegafur).Described enzyme is mainly produced by liver, and expresses (Drus Metab Dispos., 29 (2): 91-5,2001 on a small quantity in such as organs such as lung, large intestine, mammary gland, kidney and uterus; AdvDrug Deliv Rev., 18; 54 (10): 1245-56,2002).

Human cell's cytochrome p 450 2D6 (hereinafter claiming CYP2D6) gene is positioned on No. 22 karyomit(e), and pseudogene CYP2D7 and CYP2D8 are in an end of CYP2D6 gene.The known metabolism of being responsible for the important clinical medicine (comprising psychoactive drug, cardiovascular agent, morphine medicine etc.) more than 100 kinds by the enzyme of described genes encoding.

Enzyme by described CYP2D6 genes encoding is mainly produced by liver.Although described enzyme accounts for the about 2% of cytochrome P 450 enzymes total amount, they are the main enzymes that participate in 30% drug metabolism.

Described enzyme active different in individuality, and be divided into PM (weak metabolism agent), IM (medium metabolism agent), EM (strong metabolism agent) and UM (ultrafast metabolism agent) according to its active rank.Described Gene Polymorphisms has partly caused the different activities of described enzyme.As everyone knows, the CYP1A2 gene has represented genetic polymorphism.Up to the present, in promotor, exon and the intron of described CYP1A2 gene, found the varient more than 24 kinds.Till in June, 2007, existing 36 kinds of haplotypes (combination of genetic variant) ( Http:// www.cypalleles.ki.se/cyp1a2.htm), the genotype of 50 kinds of CYP2A6 ( Http:// www.cyDalleles.ki.se.cyp2a6.htm) and about 80 kinds of CYP2D6 Gene Polymorphisms ( Www.immi.ki.se.cypalleles/cyp2d6.htm), they have great difference between species.Because various types of gene variants and haplotype were in the news, therefore be necessary single nucleotide polymorphism (SNP) by minimum thus determine time of haplotype enhancing accurately and cost effectiveness.

Before various medicines are absorbed by the body and discharge, metabolism and transhipment will be carried out always.Cytochrome P450 (CYP) and drug transporter participate in metabolism and transhipment.Research to the CYP enzyme that participates in drug metabolism is being carried out always energetically.At present, 15 kinds of CYP enzyme, especially CYP2D6, CYP2C9, CYP3A4, CYP2B6, MDR1 and CYP2C19, being in the news has genetic polymorphism.Described genetic polymorphism is the principal element of clinical effectiveness, result of treatment and side effect of the medicine substrate of the described enzyme of influence.Some genetic variants cause the deposition of enzyme and energy metabolism medicine not.Other genetic variant partly reduces enzymic activity.For example, according to the different of described genetic variant and different on phenotype, and has relative higher similarity between genotype and the phenotype such as enzymes such as CYP2D6 and CYP2C9.Simultaneously, be difficult to whether predict the phenotype of CYP3A4, CYP2B6 and MDR1 gene according to the existence of functional genetic variant.

With regard to human, the CYP3A4 that all of metabolism 50% are taken medicine demonstrates great activity difference between individuality.Known CYP2B6 shows maximum 270 times difference between individuality.Although individual difference is so big, activity difference between the individuality still is difficult to directly predicts from genotype, because the protein expression that has the drug metabolism enzyme of low correlation or drug transporter between genotype and the phenotype great difference occurs according to the difference of external factor.For these genes, proteic expression is regulated, but not the existence of genetic variant whether, may be the prior factor that causes the individual difference of metabolic activity.After the expression of described enzyme was induced, these enzymes were controlled oneself to generate in a large number and are improved activity.The mechanism of induced expression combines with the promotor of target gene by the outer material that will comprise drug receptor and realizes.The representative instance of described drug receptor is known pregnane X acceptor (PXR) of expressing in the NR1I2 gene.

It is reported that the expression amount of PXR is according to the difference of individuality and difference.What is interesting is, the expression amount of described receptor expression amount and drug metabolism enzyme such as CYP3A4 and CYP2B6 have high correlation (Current Drug Metabolism, 2005,6:369-383).So the expression amount difference of the described drug metabolism enzyme between the individuality depends on described PXR expression of gene amount difference or activity difference, rather than depends on varient albumen.

In this, individual PXR Gene Polymorphisms or described PXR expression of gene Study on difference have been caused concern recently.Report that although aminoacid sequence does not change, some genetic variants still cause individual difference to drug reaction, promote as the CYP3A4 that causes because of Rifampin (rifampin) in erythromycin breath test or the body is active.These PXR varients are owing to the expression variation of described target gene causes activity change.Therefore, described PXR varient may cause medicine or biomolecules activity difference in vivo, and greatly influences the individual difference of the drug interaction that medicine (couplant of PXR gene) causes.

UDP-glucuronyl transferase (UGT) is the enzyme that the catalysis glucuronic acid combines with interior endogenous material of body and exogenous material.Described UDP-glucuronyl transferase produces the glucuronic acid couplant that has toxic material such as phenol, ethanol, amine and fatty acid cpds etc., and these materials are converted into water wetted material to discharge (Parkinson A from human body through bile or urine, Toxicol Pathol., 24:48-57,1996).

It is reported that described UGT mainly is present in the endoplasmic reticulum or nuclear membrane of mesenchymal cell, and expression is also arranged in other tissue such as kidney and skin.Described UGT enzyme can roughly be divided into UGT1 and UGT2 subfamily according to the similarity of one-level aminoacid sequence.There are 9 kinds of isomer (UGT1A1 and UGT1A3～UGT1A10) in human UGT1A family.Wherein, five kinds of isomer (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) are by liver expression.The genetic polymorphism of described UGT1A gene family varies with each individual.Knownly there are several genetic polymorphisms (http://galien.pha.ulaval.ca/alleles/alleles.html) with respect to UGT1A1, UGT1A3～UGT1A10 gene.The polymorphism of described UGT1A gene has great difference between the race.The activity that has confirmed enzyme is different because of the difference of polymorphism, and polymorphism is the important factor of decision for the susceptibility of pharmacological agent.UGT1A1 ^*6 and UGT1A1 ^*28 relevant with Gilbert syndrome (Monaghan G, Lancet, 347:578-581,1996).In addition, reported the various functional varient relevant with various diseases.

Owing to reported various types of genetic variants and haplotype, so searching method should be effective.Described haplotype can be analyzed by Arlequin, SNPalyze or other similar software.Analyze all single nucleotide polymorphism (SNP) and search for the heritable variation cognition of every kind of haplotype at cost and effective inadequately on the time.

In order to raise the efficiency, can provide haplotype label SNP (htSNP) system of selection.Described htSNP system of selection is a kind of method of selecting minimum set of tags to come every kind of haplotype of accurate mark.If determined selected SNP, then can predict all haplotypes.

For many genes, the distribution of genetic polymorphism is different because of race's difference.Therefore, be necessary to check whether congenital heredity varient and haplotype frequently appear at the Koryo philtrum, and if they have multifrequency numerous, and how to select htSNP according to haplotype.Yet, to the genetic variant in high beauty's the gene, corresponding haplotype and the research of selecting according to the htSNP of every kind of haplotype and few with it.

Recently, reported a kind of by the CYP2D6 genetic variant that mainly is found among the white people is carried out method (Sistonen J etc., the ClinChem.2005Jul that SNaPshot analyzes to determine 11 kinds of SNP; 51 (7): 1291-1295) and the CYP2D6 diagnosing chip of Roche or Jurilab company.Yet they concentrate on the CYP2D6 genetic variant that is found among the white people.The research of Asian CYP2D6 genetic variant that diagnosis is comprised high beauty is also not enough.

Therefore, the inventor researched and developed the human CYP1A2, the CYP2A6 that accurately determine mainly to be found in the Koryo philtrum in a kind of short period of time, CYP2D6, NR1I2 (=PXR) and the method for the varient of UGT1A gene.The invention provides to the human CYP1A2, the CYP2A6 that mainly are found in the Koryo philtrum, CYP2D6, NR1I2 (=PXR) and the htSNP system of selection of UGT1A genetic variant so that prove the operability of selected htSNP.

Summary of the invention

Therefore, one aspect of the present invention provide the CYP1A2, the CYP2A6 that are used to determine to be found in the Koryo philtrum, CYP2D6, NR1I2 (=PXR) and the htSNP system of selection of the haplotype of UGT1A gene.

In addition, another aspect of the present invention provide use described htSNP determine human CYP1A2, CYP2A6, CYP2D6, NR112 (=PXR) and the method for UGT1A gene genotype.

In addition, another aspect of the present invention also provides and has used the test kit that comprises gene chip to determine the method for human CYP2D6 gene genotype.

The others of total inventive concept and advantage part are in the following description illustrated, and will partly obviously, maybe can obtain understanding by putting into practice described total inventive concept according to described explanation.

Above-mentioned and/or others of the present invention and advantage realize that by the system of selection that the htSNP that is selected from the gene in human CYP1A2, CYP2A6, CYP2D6, PXR and the UGT1A gene is provided described method comprises: from human collection of biological sample; Extract nucleic acid in the collected sample from operation (a); Carry out PCR (polymerase chain reaction) with primer, this reaction is used the nucleic acid that extracted in the operation to increase as template and is selected from gene or its fragment in human CYP1A2, CYP2A6, CYP2D6, PXR and the UGT1A gene; Existence by the gene order definitive variation body of gained PCR product in the operation (c); Gene order by the PCR product of having determined to have varient in operation in (d) is determined haplotype; With with SNPtagger software described haplotype of analyzing in the operation (d) is checked order and selects SNP.

Above-mentioned and/or others of the present invention and advantage realize that by the method for determining human CYP2A2 gene genotype is provided described method comprises: (a) collection of biological sample from object; (b) extract genomic dna in the collected sample from operation (a); (c) carry out PCR with primer, this reaction uses the genomic dna that is extracted in the operation (b) to come amplifying human CYP1A2 gene or its fragment as template; (d) existence of at least 11 kinds of varients of definite CYP1A2 gene in the gene order of the PCR product of gained in operation (c), described at least 11 kinds of varients are selected from-3860G〉A ,-3598G〉T ,-3594T〉G ,-3113G〉A ,-2847T〉C ,-2808A〉C ,-2603insA ,-2467delT ,-1708T〉C ,-739T〉G ,-163C〉A, 1514G〉A, 2159G〉A, 2321G〉C, 3613T〉C, 5347C〉T and 5521A〉G.

Above-mentioned and/or others of the present invention and advantage realize that by the method that detects the varient in the CYP1A2 promoter gene is provided described method comprises: (a) collection of biological sample from object; (b) extract genomic dna in the collected sample from operation (a); (c) carry out PCR with primer, this reaction uses the middle gained genomic dna of operation (b) to come the promoter region of amplifying human CYP1A2 gene as template; (d) existence of definite CYP1A2 genetic variant in the gene order of PCR product of gained in operation (c), described CYP1A2 genetic variant comprises-3860G〉A ,-3598G T ,-3594T G ,-3113G A ,-2847T C ,-2808A C ,-2603insA ,-2467delT ,-1708T C ,-739T G and-163C A.

Above-mentioned and/or others of the present invention and advantage realize that by the method for determining human CYP2A6 gene genotype is provided described method comprises: (a) collection of biological sample from object; (b) extract nucleic acid in the collected sample from operation (a); (c) carry out PCR with primer, this reaction uses the middle gained nucleic acid of operation (b) to come amplifying human CYP2A6 gene or its fragment as template; (d) existence of definite CYP2A6 genetic variant in the gene order of the PCR product of gained in operation (c), described CYP2A6 genetic variant is selected from-48T〉G, 13G〉A, 567C〉T, 2134A〉G, 3391T〉C, 6458A〉T, 6558T〉C, 6582G〉T, 6600G〉T and 6091C〉T.

Above-mentioned and/or others of the present invention and advantage realize that by the method for determining human CYP2D6 gene genotype is provided described method comprises: (a) from human collection of biological sample; (b) extract nucleic acid in the collected sample from operation (a); (c) carry out PCR with primer, this reaction uses the nucleic acid of gained in the operation (b) to come amplifying human CYP2D6 gene or its fragment as template; (d) determine the existence of at least 11 kinds of varients in the CYP2D6 gene, described at least 11 kinds of varients comprise: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from-1028T C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; 1611T〉A; 1758G〉A; 1887insTA; 2573insC; 2988G〉A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

Above-mentioned and/or others of the present invention and advantage realize that by the method for determining the PXR gene genotype is provided described method comprises: (a) from human collection of biological sample; (b) extract nucleic acid in the collected sample from operation (a); (c) carry out PCR with primer, this reaction uses the middle gained nucleic acid of operation (b) to come amplifying human PXR gene or its fragment as template; (d) existence of the genetic variant of research PXR gene in the gene order of the PCR product of gained in operation (c), described genetic variant is selected from-25385C〉T ,-24113G〉A, 7635A〉G, 8055C〉T, 11156A〉C and 11193T〉C.

Above-mentioned and/or others of the present invention and advantage realize that by the method that the functional varient of determining the UGT1A gene is provided described method comprises: (a) from human collection of biological sample; (b) extract nucleic acid in the collected sample from operation (a); (c) use the nucleic acid that is extracted in the operation (b) to come amplifying human UGT1A gene individually as template; (d) to the described gene sequencing of amplification in the operation (c) and determine the existence of functional varient in the described UGT1A gene, described functional varient is selected from :-39 (TA) 6 in the UGT1A1 gene〉(TA) 7,211G A, 233C〉T and 686C A; 31T in the UGT1A3 gene〉C, 133C〉T and 140T〉C; 31C in the UGT1A4 gene〉T, 142T〉G and 292C〉T; 19T in the UGT1A6 gene〉G, 541A〉G and 552A〉C; 387T in the UGT1A7 gene〉G, 391C〉A, 392G＜A, 622T〉C and 701T〉C; With in the UGT1A9 gene-118T9 T10,726T G and 766G A.

Above-mentioned and/or others of the present invention realize that by the method with to the relevant polymorphism of the susceptibility of Rinotecan (irinotecan) of determining the UGT1A gene is provided described method comprises with advantage: (a) from human collection of biological sample; (b) extract nucleic acid in the collected sample from operation (a); (c) use the nucleic acid that is extracted in the operation (b) to come amplifying human UGT1A gene as template; (d) to the existence of varient in the also definite described UGT1A gene of the described human UGT1A gene sequencing of amplification in the operation (c), described varient is selected from: the 211G in the UGT1A1 gene〉A, 233C〉T and 686C〉A; 19T in the UGT1A6 gene〉G, 541A〉G and 552A〉C; With in the UGT1A9 gene-118T9 T10,726T G and 766G A.

Above-mentioned and/or others of the present invention and advantage determine that by providing with gene chip the method for human CYP2D6 gene genotype realizes that described method comprises: (a) extract gene to be studied and carry out the PCR product of multiplex PCR with the border (circumference) that obtains to comprise SNP to be identified; (b) ASPE (allele-specific primers extension) primer with the specificity base of recognition allele carries out the ASPE reaction; (c) described reaction product is mixed with gene chip; (d) analyze described gene chip.

Above-mentioned and/or others of the present invention and advantage are by being provided for determining the SNaPshot gene type test kit of CYP2D6 gene genotype and being used for determining that the gene chip with Zip coding (Zip Code) oligonucleotide chip of SNP realizes.

Biological specimen described in the present invention comprises blood, skin cells, myxocyte and the hair of object, and blood preferably.

Nucleic acid described in the present invention can comprise DNA or RNA, preferably DNA, more preferably genomic dna.

Varient described in the present invention will be described as follows.

Term among the present invention " aN〉M " or " NaM " (a is a positive number, and N and M are respectively A, C, T or G) are meant that the base N of " a " position in gene order is substituted by base M.Term " ainsN " or " adelN " (a is a positive number, and N is A, C, T or G) are that place, " a " position inserts or delete a base N in gene order.

For example, " 1548C〉T " varient is that the-1584 C base is substituted by the T base in the gene order.

" 2573insC " varient is the 2573rd C base of having inserted (adding) in gene order." 4125-4133insGTGCCCACT " varient is to have inserted 9 bases G TGCCCACT at the 4125th～4133 bit base place of human CYP2D6 gene.

" 2D6 deletion " varient is that whole human CYP2D6 gene is deleted from karyomit(e).

In addition, " 2D6 repetition " varient is to have repeated at least 2 human CYP2D6 genes in same karyomit(e).

As mentioned above, the invention provides and use optimum search to make up the functional varient relevant with the drug susceptibility of CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene or the method for polymorphism analyzed, described optimum search is made up based on the polymorphism of also not checked high beauty CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene so far.The present invention go for determining to be included on the genetics characteristic with Japanese like the physiognomy of Koryo and Chinese and high beauty in interior Asian CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene genotype.

Description of drawings

According to the following explanation to embodiment of the present invention in conjunction with the accompanying drawings, above-mentioned and/or others of the present invention will become apparent and be more readily understood, in described accompanying drawing:

Fig. 1 has illustrated the position of a kind of varient in the gene order of CYP1A2 gene of the CYP1A2 gene of determining first according to the present invention;

Fig. 2～6th, the example of the htSNP combination of the CYP1A2 gene of selecting according to the present invention;

Fig. 7～14 have illustrated the result of the varient of CYP1A2 promoter gene, described varient be the described CYP1A2 gene selected according to the present invention functional varient (here, X-axis represents to depend on the motion of primer molecule quantity, and Y-axis is represented the height at each peak)

Fig. 7 has illustrated the wild-type SNP of CYP1A2 promotor,

Fig. 8 has illustrated the CYP1A2 promotor that is arranged in heterozygosis (hetero) varient-3860G〉A (CYP1A2 ^*1C) ,-2467delT (CYP1A2 ^*1D) and-163C A (CYP1A2 ^*1F) varient, described heterozygosis varient contain a varient and a wild-type in double-stranded DNA,

Fig. 9 has illustrated the CYP1A2 promotor that is arranged in (homo) varient that isozygotys-3860G〉A (CYP1A2 ^*1C) ,-2467delT (CYP1A2 ^*1D) and-163C A (CYP1A2 ^*1F) varient, the described varient that isozygotys contains two varients in double-stranded DNA,

Figure 10 has illustrated the CYP1A2 promotor that is arranged in the heterozygosis varient-163C〉A (CYP1A2 ^*1F) and-2808A the C varient,

Figure 11 has illustrated the CYP1A2 promotor that is arranged in the varient that isozygotys-163C〉A (CYP1A2 ^*1F) varient, also illustrated be arranged in the heterozygosis varient-2467delT (CYP1A2 ^*1D) ,-and 739T〉G (CYP1A2 ^*1E) ,-3598G T ,-3113G A ,-2847T C and-1708T the C varient,

Figure 12 has illustrated the CYP1A2 promotor that is arranged in the varient that isozygotys-163C〉A (CYP1A2 ^*1F) varient, also illustrated be arranged in the heterozygosis varient-2467delT (CYP1A2 ^*1D) ,-3598G T and-2847T the C varient,

Figure 13 has illustrated the CYP1A2 promotor that is arranged in the heterozygosis varient-163C〉A (CYP1A2 ^*1F) ,-2467delT (CYP1A2 ^*1D) and-3594T the G varient,

Figure 14 has illustrated the CYP1A2 promotor that is arranged in the varient that isozygotys-163C〉A (CYP1A2 ^*1F) varient, also illustrated be arranged in the heterozygosis varient-3860G A (CYP1A2 ^*C) ,-2467delT (CYP1A2 ^*1D) and-the 2603insA varient;

Figure 15 has illustrated and has been used for selecting kind and the frequency of the CYP2A6 of htSNP combination at Koryo people's haplotype according to the present invention;

The htSNP combination of the CYP2A6 gene of selecting according to the present invention is for example understood in Figure 16～21,

Figure 16 illustrated and has been used for determining the selection that the htSNP of the haplotype of CYP2A6 gene makes up by detect 3 kinds of genetic variants that target adds 6 kinds of functional varients and have a called function at genetic variant,

Figure 17 has illustrated the selection of the htSNP combination of the haplotype that is used for definite CYP2A6 gene, and described CYP2A6 gene comprises 8 kinds and contains varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants that amino acid is replaced,

Figure 18 has illustrated the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene, described CYP2A6 gene comprises 8 kinds and contains varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants that amino acid is replaced

Figure 19 has illustrated the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene, described CYP2A6 gene comprises 8 kinds and contains varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants that amino acid is replaced

Figure 20 has illustrated the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene, described CYP2A6 gene comprises 8 kinds and contains varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants that amino acid is replaced

Figure 21 has illustrated the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene, described CYP2A6 gene comprises 8 kinds and contains varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants that amino acid is replaced

Figure 22～30 have illustrated the result to the SNaPshot analysis of selected htSNP combination and the functional genetic variant combination of CYP2A6,

Figure 22 has illustrated the CYP2A6 gene that is arranged in the heterozygosis varient-48T〉G, 2134A〉G and 6558T〉the C varient, described heterozygosis varient contains a varient and a wild-type in double-stranded DNA,

Figure 23 has illustrated the 567C of the CYP2A6 gene that is arranged in the heterozygosis varient〉7 varients, described heterozygosis varient contains a varient and a wild-type in double-stranded DNA,

Figure 24 has illustrated the 6458A of the CYP2A6 gene that is arranged in the heterozygosis varient〉T and 6558T〉the C varient, described heterozygosis varient contains a varient and a wild-type in double-stranded DNA,

Figure 25 has illustrated the CYP2A6 gene that is arranged in the heterozygosis varient-48T〉G, 13G〉A and 6558T〉the C varient, described heterozygosis varient contains a varient and a wild-type in double-stranded DNA,

Figure 26 has illustrated the 3391T of the CYP2A6 gene that is arranged in the heterozygosis varient〉the C varient, described heterozygosis varient contains a varient in double-stranded DNA, and another is deleted,

Figure 27 has illustrated the CYP2A6 gene that is arranged in the heterozygosis varient-48T〉G and 2134A〉the G varient, described heterozygosis varient contains a varient in double-stranded DNA, and another is deleted,

Figure 28 has illustrated the CYP2A6 gene that is arranged in the heterozygosis varient-48T〉G, 6558T〉C and 6600G〉the T varient, described heterozygosis varient contains a varient in double-stranded DNA, and another is deleted,

Figure 29 has illustrated the 6458A of the CYP2A6 gene that is arranged in the heterozygosis varient〉the T varient, described heterozygosis varient contains a varient in double-stranded DNA, and another is deleted,

Figure 30 has illustrated the 6558T of the CYP2A6 gene that is arranged in the heterozygosis varient〉C and 6582G〉the T varient, described heterozygosis varient contains a varient and a wild-type in double-stranded DNA;

Figure 31 and 32 has illustrated that the SNaPshot that carries out with gene studies described in Figure 22～30 analyzes, and described SNaPshot analyzes the CYP2A6 gene elmination the described genetic variant of having determined in addition in Figure 22～30,

Figure 31 has illustrated the CYP2A6 gene that is present in the homologous chromosomes,

Figure 32 has illustrated the CYP2A6 gene that is not present in the karyomit(e) and has only a gene;

Figure 33 has illustrated that the conjugation of partial C YP2A6 gene and CYP2A7 gene is connected;

Figure 34～39 have illustrated the htSNP combination of the CYP2D6 gene of selecting according to the present invention,

Figure 40 and 41 has illustrated the SNaPshot analytical results of one of htSNP combination of the CYP2D6 gene of selecting according to the present invention;

Figure 42 has illustrated and has used gene chip to determine the process of CYP2D6 gene genotype;

Figure 43 has illustrated the probe on the described gene chip that is used for the CYP2D6 gene;

Figure 44 has illustrated the CYP2D6 gene amplification of using long PCR to carry out;

Figure 45 has illustrated the ASPE reaction process;

Figure 46 has illustrated the gene chip that shows according to the analytical results of the varient in 12 pairs of CYP2D6 genes of illustrative embodiments;

Figure 47 has illustrated the htSNP combination of the PXR gene of selecting according to the present invention;

Figure 48～50 have illustrated result's (, X-axis represents to depend on the motion of the molecular amounts of each primer, and Y-axis represents the height at each peak) of the functional varient in the PXR gene that search selects according to the present invention here,

Figure 48 has illustrated PXR gene-25385C〉T ,-24113G〉A, 7635A〉G, 8055C〉T, 11156A〉C and 11193T〉the functional varient of C, described varient is wild-type;

Figure 49 has illustrated the PXR gene that is arranged in the heterozygosis varient-25385C〉T ,-24113G〉A, 7635A〉G, 8055C〉T, 11156A〉C and 11193T〉the functional varient of C, described heterozygosis varient contains a varient and a wild-type in double-stranded DNA;

Figure 50 has illustrated the PXR gene that is arranged in the varient that isozygotys-25385C〉T ,-24113G〉A, 7635A〉G, 8055C〉T, 11156A〉C and 11193T〉the functional varient of C, the described varient that isozygotys contains two varients in double-stranded DNA;

Figure 51～54 have illustrated the analytical results of the functional varient of 50 high beauties' UGT1A gene (here, X-axis is represented the position of SNP, and Y-axis is represented the height at each peak, and redness is T, and black is C, and blueness is G, and green is A);

Figure 51 has illustrated the analytical results to the functional varient of UGT1A1 (a) and UGT1A3 (b) gene;

Figure 52 has illustrated the analytical results to the functional varient of UGT1A4 (a) and UGT1A6 (b) gene;

Figure 53 has illustrated the analytical results to the functional varient of UGT1A7 gene;

Figure 54 has illustrated the analytical results to the functional varient of UGT1A9 gene;

Figure 55 has illustrated 50 high beauties' UGT1A1, UGT1A6 and UGT1A9 gene and analytical results to the relevant polymorphism of the susceptibility of Rinotecan (irinotecan);

(a) 211G of UGT1A1 gene〉A, 233C〉T and 686C〉A; The 19T of UGT1A6 gene〉G, 541A〉G and 552A〉C; And the 726T of UGT1A9 gene〉G and 766G〉A, the above-mentioned wild-type that is,

(b) the wild-type 211G of UGT1A1 gene〉A and 233C〉T, heterozygous 686C〉A; The heterozygous 19T of UGT1A6 gene〉G and 552A〉C, wild-type 541A〉G; The wild-type 726T of UGT1A9 gene〉G and 766G〉A,

(c) the wild-type 211G of UGT1A1 gene〉A, 233C〉T and 686C〉A; The heterozygous 19T of UGT1A6 gene〉G, 541A〉G and 552A〉C; The heterozygous 726T of UGT1A9 gene〉G and wild-type 766G A and

(d) the heterozygous 211G of UGT1A1 gene〉A and 233C〉T, wild-type 686C〉A; The heterozygous 19T of UGT1A6 gene〉G, 541A〉G and 552A〉C; The wild-type 726T of UGT1A9 gene〉G and 766G〉A.

Embodiment

Illustrative embodiments of the present invention hereinafter is described with reference to the accompanying drawings, and wherein identical Reference numeral is represented identical element, and will avoid duplicate explanation as far as possible.

The present invention can be for the CYP1A2 gene genotype by the varient analysis of high beauty CYP1A2 gene being determined to be found in the Koryo philtrum, select htSNP as the optimum set of tags of each haplotype and confirm its operability.In addition, the present invention can be for the new haplotype of determining human CYP1A2 gene.

The method of the htSNP of the human CYP1A2 gene of selection of the present invention is as follows:

(a) collection of biological sample from object;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the nucleic acid that is extracted in the operation (b) to come amplifying human CYP1A2 gene or its fragment as template;

(d) existence of definitive variation body in the gene order of the PCR product of gained from operation (c);

(e) determined that to operating in (d) the described PCR product with varient checks order with SNPtagger software.

The method of extracting nucleic acid (a) in the collected sample from operation is well known in the art without limits.Alternatively, can use the extraction test kit to extract described nucleic acid.For example, can use the DNA or the RNA that produce by Qiagen company (U.S.) and Stratagene company (U.S.) to extract test kit.If that extract from described test kit is RNA, it is stand-by then to produce cDNA by reverse transcription.

The fragment of human CYP1A2 gene is meant the fragment of the known varient that comprises human CYP1A2 gene described in the operation (c), for example, and single nucleotide polymorphism (SNP).Increase described human CYP1A2 gene or its segmental described primer can design based on human CYP1A2 gene or its segmental gene order, and can be selected from the primer that contains with reference to 2～31, but is not limited thereto.

Described varient in the operation (d) comprises SNP, gene elmination and gene redundancy, but is not limited thereto.For example, described varient can comprise 17 kinds of varients, as table 5.

Described sequence measurement can be a method known in the art without limits.For example, can use automated DNA sequenator or carry out tetra-sodium and check order to determine gene order.The order-checking of described tetra-sodium is the known SNP measuring method that is used for dna sequencing, is the method that the light of the inorganic pyrophosphate (PPi) that discharges when detecting from the DNA polymerization is expressed.Described dna sequencing can use from the primer with reference to 32～61 and carry out, but is not limited thereto.Can determine the existence of varient in the operation (d) by the gene order that compares wild-type CYP1A2 gene.Can use the gene order of described wild-type CYP1A2 gene, for example with reference to 1 gene order (gene pool (GenBank) accession number: NT_010194) or the genotypic gene order (DrugMetab.Pharnmacokinet of each CYP1A2 known in the art, 2005,20 (1): 24-33).

The frequency of haplotype and type can use the program of selling on technical program known in the art or the market to estimate.For example, can use the Haploview of distributed for free, or commercialization program SNPAlyze.Described Haploview software is known in the art.More preferably, can from Http:// www.broad.mit.edu/mpg/haploviewDownload described software.

Method of the present invention can comprise the repetition to operation (a)～(d) in addition.In order to determine variation phase and haplotype thereof, can detect the genotypic frequency of CYP1A2 and from described colony, select frequent CYP1A2 genotype executable operations (e) afterwards such as the intravital CYP1A2 gene of particular cluster such as race or patient.

In operation (e), the haplotype data of using SNPtagger software analysis CYP1A2 gene are to select htSNP.Described SNPtagger software is known in this area, for example Genehunter, Merlin, Allegro, SNPHAP, htSNP finder (based on PCA).More preferably, described software can from Http:// www.well.ox.ac.uk/～xiayi/haplotypeOr Http:// slack.ser.man.ac.uk/progs/htsnp.htmlDownload.

Can verify that thereby selected htSNP improves precision and determines two times of types.After determining human genotype, described genotype is decoded to determine two kinds of haplotype combination by double-stranded karyomit(e).If analyze several SNP simultaneously, then the combination of specific haplotype can be identical with the combination of another haplotype.If described genotype should verify then by the diagnosis decoding of being developed according to the present invention whether it has accurately determined described genotype.(Mai Siwoke softcom limited (TheMathWorks, Inc.), the U.S.) analyzes and whether by the genetic analysis result described genotype has been carried out correct decoding, thereby carries out described checking can to use Matlab.

In an exemplary embodiment of the present invention, at first studied the genotypic htSNP of CYP1A2 that varient in the high beauty CYP1A2 gene selects to be found in the Koryo philtrum.As a result, in high beauty CYP1A2 gene, find 17 SNP (referring to table 5) altogether.Having one among 17 SNP (is novel 2603insA).

The described single SNP that the present invention provides first comprises a varient and a wild-type (referring to Fig. 1) in double-stranded DNA.

In another illustrative embodiments of the present invention, determined to be found in the haplotype of 17 SNP of Koryo philtrum.As a result, the present invention determined the CYP1A2 gene never in people from Koryo, found before this haplotype (referring to table 6) and based on the genotype of above-mentioned haplotype.For example, the haplotype 2 (CYP1A2 of the CYP1A2 gene in the table 6 ^*1L) be meant that there is the genotype of SNP in the gene order of described CYP1A2 gene-3860 ,-2467 and-163 bit base places.More specifically, described genotype has-3860G〉A ,-2467T delT (2467delT) and-163C the SNP of A.

In another illustrative embodiments of the present invention, the haplotype of having been determined by the gene order that contains the varient in the CYP1A2 gene with the SNPtagger software analysis is so that select htSNP, that is is found in the minimum mark of 17 haplotypes of varient of the CYP1A2 gene of Koryo philtrum.The example of the htSNP combination of selecting according to the present invention is as shown in Fig. 2～6.

The described htSNP combination of selecting according to the present invention can be used for determining the haplotype of human CYP1A2 gene.Therefore, the invention provides the method for the haplotype of determining human CYP1A2 gene.Described method comprises the steps:

(a) collection of biological sample from object;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the middle gained nucleic acid of operation (b) to come amplifying human CYP1A2 gene or its fragment as template; With

(d) existence of varient in definite described CYP1A2 gene in the gene order of the PCR product of gained in operation (c), described varient is selected from-3860G〉A ,-3598G〉T ,-3594T〉G ,-3113G〉A ,-2847T〉C ,-2808A〉C ,-2603insA ,-2467delT ,-1708T〉C ,-739T〉G ,-163C〉A, 1514G〉A, 2159G〉A, 2321G〉C, 3613T〉C, 5347C〉T and 5521A〉G.

The method of the described extraction nucleic acid in the operation (b) is same as described above.

As mentioned above, the fragment of described human CYP1A2 gene is meant the fragment of the known SNP that comprises described human CYP1A2 gene.The described primer that is used for operation (c) can be selected from without limits with reference to 2～31.

The described SNP of research can be selected from the htSNP in Fig. 4～6 in the operation (d).The existence of described SNP can be studied by following varient: among Fig. 4-3860G〉A ,-3598G〉T ,-3113G〉A ,-2808A〉C ,-2603msA ,-2467delT ,-163C〉A, 1514G〉A, 2159G〉A, 5347C〉T and 5521A〉G; Among Fig. 5-3860G〉A ,-3113G〉A ,-2808A〉C ,-2603insA ,-2467delT ,-739T〉G ,-163C〉A, 1514G〉A, 2159G〉A, 5347C〉T and 5521A〉G; And among Fig. 6-3860G〉A ,-3598G〉T ,-3594T〉G ,-3113G〉A ,-2808A〉C ,-2603insA ,-2467delT ,-163C〉A, 1514G〉A, 2159G〉A, 5347C〉T and 5521A〉G, or-3860G〉A ,-3598G〉T, 2321G〉C ,-3113G〉A ,-2808A〉C ,-2603insA ,-2467delT ,-163C〉A, 1514G〉A, 2159G〉A, 5347C〉T and 5521A〉G.

The described SNP that detects in the operation (d) is based on the varient in the CYP1A2 gene that is found in the Koryo philtrum, and this SNP has specificity to the haplotype and the genotype of the CYP1A2 gene of determining high beauty.

The SNP of the CYP1A2 gene in the gene order of the described PCR product in the operation (d) can determine with polymorphism analyzing method known in the art.Preferably, described SNP can be analyzed (referring to [Peter M.Vallone etc., Int J Legal Med, 2004,118:147-157]), electrophoretic analysis or their combination by SNaPshot and determine, more preferably analyzes to determine with SNaPshot.

Described SNaPshot analysis is meant by carry out PCR with primer reacts to determine that genotypic method, described primer contain near annealing gene order the SNP site (except that the SNP zone) and ddNTP.The described SNaPshot that uses among the present invention designs and makes with the SNP of currently known methods based on the described CYP1A2 gene of research in the operation (c).Used SNaPshot is variable, as long as its base that contains next-door neighbour SNP site comprises the annealing gene order adjacent with the SNP site as 3 ' end, and has added the T base at 5 ' end and gets final product.More specifically, primer can be selected from reference to 64～74.Preferably, the length of described the anneal gene order adjacent with the SNP site is approximately 20bp.If determine several SNP simultaneously, the length of the T base of then described SNaPshot primer 5 ' end is designed to variable.For example, add 5 T bases so that the varying in size of primer, thereby change the length of described PCR product at described 5 ' end.Then, with described SNaPshot primer with and each SNP complementary ddNTP combine.Described composition varies in size because of the difference of SNP.Therefore, can determine several SNP simultaneously.

Whether correct for determining with the gene type result of described SNaPshot analysis gained, carried out the another kind of described genotypic method of determining.Another kind method preferably includes automated DNA order-checking or tetra-sodium order-checking without limits.

11 SNP that are found in 17 kinds of varients in the described CYP1A2 gene of Koryo philtrum are positioned at promoter region.Described 11 SNP comprise by the present invention determine first-the 2603insA varient.Therefore, the invention provides the method for determining varient in the human CYP1A2 promoter gene.Described method comprises the following steps:

(a) collection of biological sample from object;

(b) extract genomic dna in the collected sample from operation (a);

(c) use primer to carry out PCR, the promoter region of the genomic dna of gained in operation (b) as the human CYP1A2 gene of template amplification used in described reaction; With

(d) existence of varient in definite described CYP1A2 gene in the gene order of PCR product of gained in operation (c), described varient comprises-3860G〉A ,-3598G T ,-3594T G ,-3113G A ,-2847T C ,-2808A C ,-2603insA ,-2467delT ,-1708T C ,-739T G and-163C A.

The method of the described extraction genomic dna in the operation (b) is same as described above.

The described primer of promoter region of amplifying human CYP1A2 gene is variable in the operation (c), if described primer can increase from reference 1 from-3860G A is to-163C the SNP of A, more preferably the SNP from reference 62 and 63 gets final product.

The described SNP of the CYP1A2 gene described in the operation (d) in the gene order of PCR product can determine with polymorphism analyzing method well known in the art.Preferably, described SNP can analyze with SNaPshot and determine.The used SNaPshot analysis of the present invention can use the primer that designs based on described 11 SNP of CYP1A2 gene to carry out.The used SNaPshot primer of the present invention is variable, as long as it is designed to comprise and the adjacent gene order of sequence except that described SNP.More preferably, the primer that has the gene order of reference of being selected from 64～74.

In another illustrative embodiments of the present invention, based on 11 SNP in the described promoter region that influences the CYP1A2 enzymic activity, by the varient of the described CYP1A2 promoter gene of SNaPshot analyzing and testing.As a result, confirm described method of the present invention varient (referring to Fig. 7～14) in the described CYP1A2 promoter gene of high speed detection exactly.

2.CYP2A6

The present invention can be for the CYP2A6 gene genotype by the varient analysis of high beauty CYP2A6 gene being determined mainly to be found in the Koryo philtrum, selection htSNP as the optimum set of tags of each haplotype and confirm its operability.

Select the method for htSNP of human CYP2A6 gene as follows according to the present invention:

(a) collection of biological sample from object;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the nucleic acid that is extracted in the operation (b) to come amplifying human CYP2A6 gene or its fragment as template;

(d) determine the existence of varient in the gene order of PCR product of gained in operation (c);

(e) determine haplotype by the gene order of the PCR product of having determined to have varient in operation in (d); With

(f) with SNPtagger software ( Http:// www.well.ox.ac.uk/～xiayi/haplotyDe/) the described haplotype of determining in the operation (e) is checked order to select htSNP.

The method of extraction nucleic acid is the currently known methods in this area without limits in the described sample of collecting from operation (a).Alternatively, can use the extraction test kit to extract described nucleic acid.For example, can use the DNA or the RNA that produce by Qiagen company (U.S.) and Stratagene company (U.S.) to extract test kit.If that extract is RNA, it is stand-by then to produce cDNA by reverse transcription.

The fragment of human CYP2A6 gene is meant the fragment of the known varient that comprises human CYP2A6 gene described in the operation (c), for example, and single nucleotide polymorphism (SNP).Increase described human CYP2A6 gene or its segmental described primer can design based on human CYP2A6 gene or its segmental gene order, and can be selected from the primer with reference to 76～89, but is not limited thereto.

Described varient in the operation (d) comprises SNP, gene elmination and gene redundancy, but is not limited thereto.For example, described varient can comprise 30 kinds of varients, as table 15.

The method of the existence of definitive variation body can comprise varient detection method known in the art in the operation (d).Preferably, come the gene order of wild-type CYP2A6 gene more known in the art (for example with reference to 75 gene order (gene pool accession number: NC_000019)) or genotypic each gene order of CYP2A6 known in the art with sequential analysis or electrophoretic analysis.In addition, also available rflp analysis comes the cutting phase of the Restriction Enzyme of more described wild-type CYP2A6 gene.The deletion of described CYP2A6 gene or repetition can be by determining the electrophoretic analysis of PCR product.Described order-checking can be undertaken by automated DNA sequenator or tetra-sodium order-checking.

The described haplotype that operation is determined in (e) to contain in the gene order of PCR product of varient can be by determining such as SNPAlyze, Haplotyper, Arlequin supervisor.

Method of the present invention can comprise the repetition to operation (a)～(d) in addition.In order to determine variation phase, can detect the genotypic frequency of CYP2A6 and from described colony, select frequent CYP2A6 genotype executable operations (f) afterwards such as the intravital CYP2A6 gene of particular cluster such as race or patient.

In operation (f), the gene order of operating the described haplotype of determining in (e) with the SNPtagger software analysis is to select htSNP.Be used to select the described software of htSNP to comprise HapBlock, LDSelect, Haploview, htSNP, TagIT and tagSNPs and SNPtagger.Described SNPtagger software is known in this area, and preferably can from Http:// www.well.ox.ac.uk/～xiayi/haplotypeDownload.Can verify that thereby selected htSNP improves precision and determines two times of types.Can use Matlab (Mai Siwoke softcom limited, the U.S.) to verify described htSNP.

In an exemplary embodiment of the present invention, at first studied the genotypic htSNP of CYP2A6 that varient in the CYP2A6 gene that is found in the Koryo philtrum is selected high beauty.As a result, in high beauty CYP2A6 gene, find 30 SNP (referring to table 15) altogether.

In another illustrative embodiments of the present invention, the SNPAlyze that produces with DYNACOM company has determined the haplotype of 14 SNP among selected 30 SNP, thereby has determined to have 19 haplotypes altogether of the frequency more than 1%.Described 14 SNP comprise 8 varient and 6 frequent varients of causing amino acid to replace and contain functional genetic variant.Be used for determining that the program of described haplotype is not limited to described SNPAlyze.Alternatively, can use various softwares known in the art, for example, Haplotyper (http://www.people.fas.harvard.edu/～junliu/Haplo/docMain.htm), Arlequin (htt: //lgb.unife.ch/arlequin) and the SNP Analyzer (http://www.istech21.com/) that produces of Istech company.

In another illustrative embodiments of the present invention, with the SNPtagger software analysis comprise that the gene order of 20 haplotypes of 19 haplotypes and 1 gene elmination and frequency to select htSNP, also can identify the minimum mark of the CYP2A6 gene genotype that mainly is found in the Koryo philtrum easily.The example of htSNP is shown in Figure 16～21.

Can use the selected htSNP of the present invention to make up to determine described human CYP2A6 gene genotype.Therefore, the invention provides the method for determining described human CYP2A6 gene genotype.Described method comprises the steps:

(a) collection of biological sample from object;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the nucleic acid that is extracted in the operation (b) to come amplifying human CYP2A6 gene or its fragment as template;

(d) existence of varient in definite described CYP2A6 gene in the gene order of the PCR product of gained in operation (c), described varient is selected from-48T〉G, 13G〉A, 567C〉T, 2134A〉G, 3391T〉C, 6458A〉T, 6558T〉C, 6582G〉T, 6600G〉T and 6091C〉T.

The method of the described extraction nucleic acid in the operation (b) is same as described above.

As mentioned above, the fragment of described human CYP2A6 gene is meant the fragment of the known varient that comprises human CYP2A6 gene, for example, and single nucleotide polymorphism (SNP).The primer that can be used for operation (c) can comprise the primer of reference 90,91,120 and 130, but is not limited thereto.

Described varient in the operation (d) can be selected from the htSNP in Figure 16～21.For example, the operation (d) in, described varient can by among Figure 16-48T G; 22C〉T; 567C〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; 6582G〉T; 6600G〉T; Be selected from 6091C〉T, 5971G〉A and 5983T〉a kind of among the G; And 13G〉A; 51G〉A; 1620T〉C; And 1836G〉T determines.Described varient also can by among Figure 17-48T G; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 3391T〉C; 6458A〉T; 6558T〉C; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G determine.

As shown in figure 18, described varient can be by 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 3391T〉C; 6354T〉C; 6458A〉T; 6558T〉C; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G determine.

As shown in figure 19, described varient can be by-48T〉G; 13G〉A; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; Be selected from 6091C T, 5971G A and 5983T a kind of among the G determine.

As shown in figure 20, described varient can be by-48T〉G; 13G〉A; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 3391T〉C; 6458A〉T; 6558T〉C; Be selected from 6091C T, 5971G A and 5983T a kind of among the G determine.

In addition, as shown in figure 21, described varient can be by-48T〉G; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G determine.Preferably, described varient comes to determine as shown in figure 16.

In the varient in Figure 16, confirm to have varient functional or that have a very high potential function and comprised the varient of replacing amino acid or causing gene elmination.Therefore, detect the functional CYP2A6 varient in the varient among Figure 16, the described amino acid that can study in the varient among Figure 16 is replaced varient or gene elmination varient.Gene elmination almost can't be detected by SNP.When the described varient of search is deleted with marker gene, found 6091C〉the T varient.Described 6091C〉the T varient is the SNP that is found in specifically in the karyomit(e) of having deleted the CYP2A6 gene in operation (c) the institute amplification PCR products, and described 6091C〉the T varient can be used as gene elmination mark varient.Be used for determining that functional selected varient combination comprises 10 varients, promptly-48T〉G; 13G〉A; 567C〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; 6582G〉T; 6600G〉T; And 6091C〉T.5971G in the described CYP2A6 gene〉A and 5983T〉the G varient can substitute described 6091C〉the T varient comes the marker gene deletion.

The described varient of research is based on the varient in the CYP2A6 gene that mainly is found in the Koryo philtrum in the operation (d).Therefore, it has specificity to the haplotype and the genotype of definite high beauty's CYP2A6 gene.

The varient of the CYP2A6 gene in the gene order of the described PCR product in the operation (d) can be studied with polymorphism analyzing method known in the art.Can analyze (referring to [Peter M.Vallone etc., Int J Legal Med, 2004,118:147-157]), electrophoretic analysis or their combination with SNaPshot, more specifically, can use SNaPshot to analyze and study described varient.

Described SNaPshot analysis is meant by carry out PCR with primer reacts to determine that genotypic method, described primer contain near anneal sequence the SNP site (except that the SNP zone) and ddNTP.The described SNaPshot that uses among the present invention uses the currently known methods based on the SNP of the described CYP2A6 gene of research in the operation (d) to design and make.Used SNaPshot is variable, as long as its base that contains next-door neighbour SNP site comprises and the adjacent annealing gene order in described SNP site as 3 ' end, and has added the T base at 5 ' end and gets final product.More preferably, primer can be selected from reference to 97～102.Preferably, the length of described the anneal gene order adjacent with the SNP site is approximately 20bp.If determine several SNP simultaneously, the length of the T base of 5 ' end of then described SNaPshot primer is designed to variable.For example, add 5 T bases so that the varying in size of primer, thereby change the length of described PCR product at described 5 ' end.Then, with described SNaPshot primer with and each SNP complementary ddNTP combine.Described composition varies in size because of the difference of described SNP.Therefore, can determine several SNP simultaneously.

Then, amplification can be analyzed with the known sequence measurement in this area with the gene order of the described PCR product that is used for SNaPshot and analyzes, preferably uses the automated DNA order-checking to analyze.

For example, have and be selected from the htSNP combination that can in operation (c), be used for studying Figure 16 with reference to the primer of 92～101 gene order.Preferably, can use all primers, but be not limited thereto from reference 92～101.

Then, amplification can be analyzed with the known sequence measurement in this area with the gene order of the described PCR product that is used for SNaPshot and analyzes, preferably uses the automated DNA order-checking to analyze.

In another illustrative embodiments of the present invention, the operability of selected htSNP combination is confirmed.By from the htSNP of Figure 16 combination, selecting 10 kinds of functional or potential function CYP2A6 varients to come the gene order of the PCR product of gained in the analysis operation (c), analyze thereby carry out SNaPshot.As a result, confirm that method of the present invention can determine to be found in the CYP2A6 genotype (referring to Figure 22～32) of Koryo philtrum at high speed simultaneously.

Can comprise-48T with the described CYP2A6 gene genotype that the method for the invention is determined G, 13G A, 567C T, 2134A G, 3391T C, 6458A T, 6558T C, 6582G T, 6600G T and 6091C T.Corresponding with it each genotype and varient are as shown in Figure 22～32.For example, Figure 22 has illustrated and has contained-48T〉G, 6558T〉C and 2134A〉genotype of G varient and 7 wild-types.Figure 23 has illustrated and has contained 567C〉genotype of T and 9 wild-types.Described CYP2A6 ^*4 genotype comprise 2A6 deletion varient.Behind deletion CYP2A6 gene from human chromosomal, can not generate enzyme.If the CYP2A6 gene is deleted, then described gene is shaped as the shape that portion C YP2A6 gene and portion C YP2A7 gene combine.Described deletion specificity varient can above-mentionedly be determined in conjunction with gene by studying.

3.CYP2D6

The present invention can be for the CYP2D6 gene genotype by the varient analysis of high beauty CYP2D6 gene being determined mainly to be found in the Koryo philtrum, selection htSNP as the optimum set of tags of each haplotype and confirm its operability.

Select the method for htSNP of human CYP2D6 gene as follows according to the present invention:

(a) collection of biological sample from the mankind;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the nucleic acid that is extracted in the operation (b) to come amplifying human CYP2D6 gene or its fragment as template;

(d) by the existence of gene order definitive variation body of the PCR product of gained in operation (c);

(e) determine haplotype by the gene order of the PCR product of having determined to have varient in operation in (d); With

(f) with SNPtagger software determined haplotype in the operation (e) is checked order to select htSNP.

The method of extraction nucleic acid is the currently known methods in this area without limits in the described sample of collecting from operation (a).Alternatively, can use the extraction test kit to extract described nucleic acid.For example, can use the DNA or the RNA that produce by Qiagen company (U.S.) and Stratagene company (U.S.) to extract test kit.If that extract is RNA, it is stand-by then to produce cDNA by reverse transcription.

The fragment of human CYP2D6 gene is meant the fragment of the known varient that comprises human CYP2D6 gene described in the operation (c), for example, and single nucleotide polymorphism (SNP).Increase described human CYP2D6 gene or its segmental described primer can design based on human CYP2D6 gene or its segmental gene order.For example, described primer can comprise the gene order that is selected from reference 106, reference 107, reference 121～127, reference 129～136, reference 138, reference 139, reference 140 and reference 150, but is not limited thereto.

Described varient in the operation (d) comprises SNP, gene elmination and gene redundancy, but is not limited thereto.For example, described varient can comprise 33 kinds of varients, as table 34.

The method of the existence of definitive variation body can comprise varient detection method known in the art in the operation (d).Preferably, can determine described varient with gene sequencing, electrophoretic analysis and rflp analysis.Described gene sequencing can be undertaken by automated DNA sequenator or tetra-sodium order-checking.The order-checking of described tetra-sodium is the known SNP measuring method that is used for dna sequencing, is the method that the light of the inorganic pyrophosphate (PPi) that discharges when detecting from the DNA polymerization is expressed.

Can determine the existence of the varient in the operation (d) by the gene order that compares wild-type CYP2D6 gene.The gene order of described wild-type CYP2D6 gene is known in the art.For example, can use with reference to 105 gene order (the gene pool accession number: AY545216) or the genotypic gene order of each CYP2D6 known in the art (gene pool accession number: M33388, Http:// www.cypalleles.ki.se/cyp2d6.htm).In addition, also can carry out the cutting phase that rflp analysis comes the Restriction Enzyme of more described wild-type CYP2D6 gene.The deletion of described CYP2D6 gene or repetition can be by determining the electrophoretic analysis of PCR product.

The haplotype that operation has been determined in (d) to contain in the gene order of PCR product of varient can be determined with full order-checking.

Method of the present invention can comprise the repetition to operation (a)～(d) in addition.In order to determine variation phase, can detect the genotypic frequency of CYP2D6 and from described colony, select frequent CYP2D6 genotype executable operations (f) afterwards such as the intravital CYP2D6 gene of particular cluster such as race or patient.

In operation (f), the gene order of operating the haplotype of determining in (e) with the SNPtagger software analysis is to select htSNP.Described SNPtagger software is known in this area, for example Genehunter, Merlin, Allegro, SNPHAP, htSNP finder (based on PCA), more preferably, described software can from Http:// www.well.ox.ac.uk/～xiayi/haplotypeOr Http:// slack.ser.man.ac.uk/progs/htsnp.htmlDownload.

Can verify that thereby selected htSNP improves precision and determines two times of types.After determining human genotype, described genotype is decoded to determine two kinds of haplotype combination by double-stranded karyomit(e).If determine several SNP simultaneously, then the combination of specific haplotype may be identical with the combination of another haplotype.If described genotype should verify then by the diagnosis decoding of being developed according to the present invention whether it has accurately determined described genotype.Can use Matlab (Mai Siwoke softcom limited, the U.S.) to analyze whether described genotype has been carried out correct decoding, thereby carry out described checking by the genetic analysis result.

In an exemplary embodiment of the present invention, at first studied varient in the CYP2D6 gene that is found in the Koryo philtrum to select the genotypic htSNP of high beauty CYP2D6.As a result, 33 kinds of varients and corresponding with it 12 haplotypes (genotype) (referring to table 34 and 35) in high beauty CYP2D6 gene, have been found.

In another illustrative embodiments of the present invention, with SNPtagger software 12 CYP2D6 genotype are checked order to select htSNP, also can identify the genotypic minimum mark of the CYP2D6 that mainly is found in the Koryo philtrum easily.The example of the htSNP combination of selecting according to the present invention is as shown in Figure 34～39.

The described htSNP combination of selecting according to the present invention can be used for determining human CYP2D6 gene genotype.Therefore, the invention provides the method for determining human CYP2D6 gene genotype.Described method comprises the steps:

(a) collection of biological sample from the mankind;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the nucleic acid that is extracted in the operation (b) to come amplifying human CYP2D6 gene or its fragment as template;

(d) existence of at least 11 kinds of varients in definite CYP2D6 gene in the gene order of PCR product of gained in operation (c), described at least 11 kinds of varients comprise: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; 1611T〉A; 1758G〉A; 1887insTA; 2573insC; 2988G〉A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

The method of the described extraction nucleic acid in the operation (b) is same as described above.

As mentioned above, the fragment of described human CYP2D6 gene is meant the fragment of the known varient that comprises human CYP2D6 gene, for example, and single nucleotide polymorphism (SNP).The primer that can be used for operation (c) can comprise the gene order that is selected from reference 106 and 107, reference 121～127, reference 129～136, reference 138,139,149 and 150.

Described varient in the operation (d) can be selected from the htSNP in Figure 34～39.For example, as shown in figure 34, can determine the existence of following varient, described varient comprises: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; 1611T〉A; 1758G〉A; 1887insTA; 2573insC; 2988G〉A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

As shown in figure 35, can determine the existence of following varient, described varient comprises :-1584C〉G; Be selected from-1426C T, 100C T and 1039C a kind of among the T; 1611T〉A; 1758G〉A; 2573insC; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; Be selected from-1245insGA ,-1028T〉C ,-377A〉C, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

In addition, as shown in figure 36, can determine the existence of following varient, described varient comprises: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T;-1584C〉G; Be selected from-1028T C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; 1611T〉A; 1758G〉A; 1887insTA; 2573insC; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

In addition, as shown in figure 37, can determine the existence of following varient, described varient comprises :-1584C〉G; Be selected from-1426C T, 100C T and 1039C a kind of among the T; 1611T〉A; 1758G〉A; 2573insC; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; Be selected from-1245insGA ,-1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; 4125-4133insGTGCCCACT;-1235A〉G; 1887insTA; The 2D6 deletion; Repeat with 2D6.

In addition, as shown in figure 38, can determine the existence of following varient, described varient comprises: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from-1028T C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; 1611T〉A; Be selected from 1661G〉C and 4180G〉a kind of among the C; 1758G〉A; 1887insTA; 2573insC; 2988G〉A; 4125-4133insGTGCCCACT;-1235A〉G; 1887insTA; The 2D6 deletion; Repeat with 2D6.

In addition, as shown in figure 39, can determine the existence of following varient, described varient comprises :-1584C〉G; Be selected from-1426C T, 100C T and 1039C a kind of among the T; 1611T〉A; 1758G〉A; 2573insC; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; Be selected from-1245insGA ,-1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; 1887insTA; 2988G〉A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

Preferably, can determine the existence of the varient among Figure 34.The described varient of determining in the operation (d) is based on the varient in the CYP2D6 gene that mainly is found in the Koryo philtrum.Therefore, it has specificity to the haplotype and the genotype of definite high beauty's CYP2D6 gene.

The varient of the CYP2D6 gene described in the operation (d) in the gene order of PCR product can be determined with polymorphism analyzing method known in the art.Preferably, can analyze (referring to [Peter M.Vallone etc., Int J Legal Med, 2004,118:147-157]), electrophoretic analysis or their combination with SNaPshot and determine described varient.If the varient among the described CYP2D6 comprises SNP, then can use SNaPshot to analyze.

Described SNaPshot analysis is meant by carry out PCR with primer reacts to determine that genotypic method, described primer contain near annealing gene order the SNP site (except that the SNP zone) and ddNTP.It is to use the currently known methods based on the SNP that operates the described CYP2D6 gene of determining in (c) to design and make that the SNaPshot that uses among the present invention analyzes.Used SNaPshot is variable, as long as its base that contains next-door neighbour SNP site comprises and the adjacent annealing gene order in described SNP site as 3 ' end, and has added the T base at 5 ' end and gets final product.Preferably, the length of described the anneal gene order adjacent with the SNP site is approximately 20bp.If determine several SNP simultaneously, the length of the T base of then described SNaPshot primer 5 ' end is designed to variable.For example, add 5 T bases so that the varying in size of primer, thereby change the length of described PCR product at described 5 ' end.Then, with described SNaPshot primer with and each SNP complementary ddNTP combine.Described composition varies in size because of the difference of described SNP.Therefore, can determine several SNP simultaneously.

For example, have and be selected from reference to 141～148 and the htSNP combination that can in operation (c), be used for studying Figure 34 with reference to the primer of 152 and 153 gene order.More preferably, can use all primers of the gene order with reference of being selected from 141～148 and reference 152 and 153.Then, can analyze with the known sequence measurement by the gene order of the analysing amplified PCR product of described SNaPshot.Described gene order surveying method can be chosen in the currently known methods in this area flexibly, preferably comprises the automated DNA order-checking.

In another illustrative embodiments of the present invention, the operability of the selected htSNP combination according to the present invention is confirmed.After described SNaPshot analysis is carried out in htSNP combination in using Figure 34, analyzed the gene order of gained PCR product.As a result, confirm that method of the present invention can determine to be found in the CYP2D6 genotype (referring to Figure 40 and 41) of Koryo philtrum at high speed simultaneously.

Can comprise CYP2D6 with the described CYP2D6 gene genotype that the method for the invention is determined ^*1A, CYP2D6 ^*2A, CYP2D6 ^*5, CYP2D6 ^*2N, CYP2D6 ^*10B, CYP2D6 ^*14B, CYP2D6 ^*18, CYP2D6 ^*21B, CYP2D6 ^*41A, CYP2D6 ^*49, CYP2D6 ^*52 and CYP2D6 ^*60.Each genotype and corresponding with it varient are as shown in table 34.Referring to table 34, for example, described CYP2D6 ^*The 1A genotype comprises a wild-type, and described CYP2D6 ^*The 2A genotype comprises in the gene order of wild-type CYP2D6 gene the varient in SNP 1, SNP 5, SNP 8, SNP 9, SNP 12～SNP 18, SNP 21, SNP 25 and SNP 28 sites.Described CYP2D6 ^*5 genotype comprise 2D5 deletion varient.After the CYP2D6 gene is deleted fully, no longer produce enzyme from human chromosomal.Described CYP2D6 ^*The 2N genotype comprises 2D6 and repeats varient.Promptly in same karyomit(e), there are 2 CYP2D6 genes at least.

The invention provides the method for using gene chip to determine human CYP2D6 gene genotype.Described method comprises the steps:

(a) extract gene to be studied, described gene is carried out multiplex PCR and obtains comprising the PCR product on the border of SNP;

(b) carry out the ASPE reaction to identify allelic specificity base with ASPE (allele-specific primers extension) primer;

(c) described reaction product is mixed mutually with gene chip; With

(d) analyze described gene chip.

The invention provides the gene type assay chip (referring to Figure 42) that is used for determining SNP, described chip contains the chip based on Zip coding (Zip Code) oligonucleotide.

Each SNP is generated a pair of primer to carry out the ASPE reaction in operation (b).The ASPE primer that is generated be 3 ' end comprise the SNP site and with allele-specific bonded gene order.Described ASPE primer comprises the Zip coding,, has the oligonucleotide of 24bp towards 5 ' end that is.The Zip that generates is coded in and contains different gene orders in each allelotrope.

The present invention has selected optimum Zip encoding sequence in disclosed gene order and the gene order with bioinformatics technique design from document, described optimum Zip encoding sequence through experimental verification not with other sample generation cross reaction.The Tm of selected sequence is 61 ℃.The Zip coding that is generated does not interfere with each other.Selected gene order has Δ G value and is the hair clip shape secondary structure more than-2.

If use described ASPE primer to carry out the ASPE reaction, 3 ' the allelic sample of holding and the described primer that then contain corresponding to described primer react so that the allele-specific extension to take place.Carry out described extension if use with the covalently bound dUTP of fluorescent material Cyanine 5 (Cy5) (Cy5-dUTP), the sample that then only contains oppositional allele can be by Cy5 fluorescent material mark (referring to Figure 45).Described fluorescent material is not limited to Cy5, can use other material, such as Cy3, TAMRA, TexasRed, Cy3.5, Rhodamin6G, SyBR Green etc.

The complementary bonded oligonucleotide probe (cZip Code, complementary Zip coding) of encoding with described Zip is provided on analysis chip of the present invention.Therefore, can discern each allelotrope (referring to Figure 43) that comprises in the sample with described Zip coding primer extension.

In described probe, the gene order of having inserted 10bp at 3 ' end is induced hybridization with target as transcribed spacer.For example, described transcribed spacer sequence preference is 5 '-CAG GCC AAGT-3 '.

Described probe of the present invention preferably comprises the gene order with reference to 158～184.

Can comprise the method known in the art in operation (c) with the method for described reaction product being mixed mutually with described gene chip and analyzing in (d) through the described chip of blended.Used DNA chip scanner can change.More preferably, the GenePix 4100B scanner that uses Axon company to produce.The picture of scanning can be analyzed with GenePix Pro 6.0 softwares.

If analyze the varient of described CYP2D6 gene with gene chip of the present invention, then its result with coming to the same thing that sequential analysis is verified.Therefore, gene chip of the present invention can the low-cost varient of analyzing range gene.

4.PXR

The htSNP that method use of the present invention is selected based on the varient in the high beauty PXR gene determines the functional varient in the PXR gene.

The method of the htSNP of the human PXR gene of selection of the present invention comprises the steps:

(a) collection of biological sample from the mankind;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the nucleic acid that is extracted in the operation (b) to come amplifying human PXR gene or its fragment as template;

(d) gained PCR product in the operation (c) is checked order existing with the definitive variation body;

(e) determine to have confirmed in the operation (d) to have haplotype in the gene order of PCR product of varient; With

(f) with SNPtagger software determined haplotype in the operation (e) is checked order and selects htSNP.

The described method of extracting nucleic acid from the sample that operation (a) is collected is the currently known methods in this area without limits.Alternatively, can use the extraction test kit to extract described nucleic acid.For example, can use the DNA or the RNA that produce by Qiagen company (U.S.) and Stratagene company (U.S.) to extract test kit.If that extract is RNA, it is stand-by then to produce cDNA by reverse transcription.

The fragment of the described human PXR gene in the operation (c) is meant the fragment of the known varient that comprises human PXR gene, for example, and single nucleotide polymorphism (SNP).Increase described human PXR gene or its segmental primer can design based on human PXR gene or its segmental gene order, and described primer can be selected from the primer with reference to 221～240, but is not limited thereto.

Varient described in the operation (d) comprises SNP, gene elmination and gene redundancy, but is not limited thereto.For example, described varient can comprise 22 kinds of varients, as table 48.

The method of the existence of definitive variation body can comprise varient detection method known in the art in the operation (d).Preferably, can carry out the existence that gene sequencing, electrophoretic analysis and rflp analysis are determined described varient.Described gene sequencing can be undertaken by automated DNA sequenator or tetra-sodium order-checking.

Can determine the existence of varient in the operation (d) by the gene order that compares wild-type PXR gene.Can use the gene order of described wild-type PXR gene, for example with reference to 2200 gene order (gene pool accession number: NT_005612) or the genotypic gene order of each PXR known in the art.In addition, also available rflp analysis comes the cutting phase of the Restriction Enzyme of more described wild-type PXR gene.The deletion of described PXR gene or repetition can be by determining the electrophoretic analysis of PCR product.

The frequency and the type that confirm to contain the haplotype in the gene order of PCR product of varient in the operation (d) can use the program of selling on technical program known in the art or the market to analyze.For example, can use the Haploview of distributed for free, or commercialization program SNPAlyze.Described Haploview software is known in the art, and more preferably, can from Http:// www.broad.mit.edu/mpg/haploviewDownload described software.

Method of the present invention can comprise the repetition to operation (a)～(e) in addition.In order to determine variation phase and haplotype thereof, can detect the genotypic frequency of PXR and from described colony, select frequent PXR genotype executable operations (f) afterwards such as the intravital PXR gene of particular cluster such as race or patient.

In operation (f), with SNPtagger software haplotype definite in the operation (e) is checked order and to select htSNP.Described SNPtagger software is known in this area, for example Genehunter, Merlin, Allegro, SNPHAP, htSNP finder (based on PCA), more preferably, described software can from Http:// www.well.ox.ac.uk/～xiavi/haplotvpeOr Http:// slack.ser.man.ac.uk/Drogs/htsnp.htmlDownload.

Can verify that thereby selected htSNP improves precision and determines two times of types.After determining human genotype, described genotype is decoded to determine two kinds of haplotype combination by double-stranded karyomit(e).If determine several SNP simultaneously, then the combination of specific haplotype may be identical with the combination of another haplotype.If described genotype should verify then by the diagnosis decoding of being developed according to the present invention whether it has accurately determined described genotype.Can use Matlab (Mai Siwoke softcom limited, the U.S.) to analyze whether described genotype has been carried out correct decoding, thereby carry out described checking by the genetic analysis result.

In an exemplary embodiment of the present invention, at first studied varient in the high beauty PXR gene to select htSNP, the functional varient of high beauty PXR gene.As a result, 22 SNP (referring to table 48) in high beauty's PXR gene, have been found altogether.

In another illustrative embodiments of the present invention, the SNPAlyze software produced with DYNACOM company has been determined the haplotype of 6 functional varients among 22 selected SNP, thereby has determined 14 haplotypes (referring to table 49) altogether.

In another illustrative embodiments of the present invention, with SNPtagger software described 14 haplotypes are checked order to select htSNP, also can determine to be found in the minimum mark (referring to Figure 47) of functional varient of the PXR gene of Koryo philtrum easily.

Can use the htSNP that selects according to the present invention to make up to determine the functional varient of human PXR gene.Therefore, the invention provides the method for the functional varient of determining human PXR gene.Described method comprises the steps:

(a) collection of biological sample from the mankind;

(b) extract nucleic acid in the collected sample from operation (a);

(c) carry out PCR with primer, this reaction uses the nucleic acid that is extracted in the operation (b) to come amplifying human PXR gene or its fragment as template; With

(d) existence of functional varient in definite PXR gene in the gene order of the PCR product of gained in operation (c), described varient is selected from-25385C〉T ,-24113G〉A, 7635A〉G, 8055C〉T, 11156A〉C and 11193T〉C.

Operation described in (b) extracted the method for nucleic acid from the sample of collecting same as described above.

The fragment of described human PXR gene is meant the fragment of the known varient that comprises human PXR gene, for example, and single nucleotide polymorphism (SNP).The primer that can be used for operation (c) can be selected from the primer of reference 242～247, but is not limited thereto.

The described SNP of research is based on the functional varient of the PXR gene that is found in the Koryo philtrum in the operation (d), and it has specificity to the functional varient of definite high beauty PXR gene and the haplotype of described functional varient.

Can determine the existence of the varient of the PXR gene in the gene order of PCR product described in the operation (d) with polymorphism analyzing method known in the art.Preferably, can analyze (referring to [Peter M.Vallone etc., Int J Legal Med, 2004,118:147-157]), electrophoretic analysis or their combination, more preferably analyze to determine the existence of described varient with SNaPshot with SNaPshot.

Described SNaPshot analysis is meant by carry out PCR with primer reacts to determine that genotypic method, described primer contain near annealing gene order the SNP site (except that the SNP zone) and ddNTP.It is to use the currently known methods based on the SNP of the described PXR gene of research in the operation (d) to design and make that the SNaPshot that uses among the present invention analyzes.Used SNaPshot is variable, as long as its base that contains next-door neighbour SNP site comprises and the adjacent annealing gene order in described SNP site as 3 ' end, and has added the T base at 5 ' end and gets final product.More preferably, primer can be selected from the primer with reference to 242～2457.Preferably, the length of described the anneal gene order adjacent with the SNP site is approximately 20bp.If determine several SNP simultaneously, the length of the T base of then described SNaPshot primer 5 ' end is designed to variable.For example, add 5 T bases so that the varying in size of primer, thereby change the length of described PCR product at described 5 ' end.Then, with described SNaPshot primer with and each SNP complementary ddNTP combine.Described composition varies in size because of the difference of described SNP.Therefore, can determine several SNP simultaneously.

Whether correct in order to determine the gene type result who uses described SNaPshot to analyze, adopted another kind of methods of genotyping.Another kind of methods of genotyping preferably includes automated DNA order-checking or tetra-sodium order-checking without limits.

In another illustrative embodiments of the present invention, the operability of the selected htSNP combination of the present invention is confirmed.Use the htSNP combination among Figure 47 to carry out described SNaPshot analysis, and analyzed the gene order of gained PCR product.As a result, confirm that method of the present invention can determine to be found in the PXR gene function varient (referring to Figure 48～50) of Koryo philtrum at high speed simultaneously.

Can comprise-25385C with the functional varient of the definite described PXR gene of method of the present invention T ,-24113G〉A, 7635A〉G, 8055C〉T, 11156A〉C and 11193T〉C.

5.UGT1A

The method of determining the functional varient of human UGT1A gene according to the present invention comprises the steps:

(a) collection of biological sample from the mankind;

(b) extract nucleic acid in the collected sample from operation (a);

(c) use individually amplifying human UGT1A gene of the nucleic acid that extracted in the operation (b); With

(d) the described gene of amplification in the operation (c) is checked order and the existence of the functional varient of definite UGT1A gene, described varient is selected from :-39 (TA) 6 in the UGT1A1 gene〉(TA) 7,211G A, 233C〉T and 686C A; 31T in the UGT1A3 gene〉C, 133C〉T and 140T〉C; 31C in the UGT1A4 gene〉T, 142T〉G and 292C〉T; 19T in the UGT1A6 gene〉G, 541A〉G and 552A〉C; 387T in the UGT1A7 gene〉G, 391C〉A, 392G＜A, 622T〉C and 701T〉C; With in the UGT1A9 gene-118T9 T10,726T G and 766G A.

The method with to the relevant polymorphism of the susceptibility of Rinotecan of definite UGT1A gene of the present invention comprises the steps:

(a) collection of biological sample from the mankind;

(b) extract nucleic acid in the collected sample from operation (a);

(c) use individually amplifying human UGT1A gene of the nucleic acid that extracted in the operation (b); With

(d) the described gene that increases in (c) checks order and the existence of definite UGT1A genetic variant to operating, and described varient is selected from: the 211G in the UGT1A1 gene〉A, 233C〉T and 686C〉A; 19T in the UGT1A6 gene〉G, 541A〉G and 552A〉C; With in the UGT1A9 gene-118T9 T10,726T G and 766G A.

Method of the present invention has adopted the optimum polymorphism set of tags of selecting based on the polymorphism of the UGT1A gene that mainly is found in high beauty, and has determined functional varient or drug susceptibility in the UGT1A gene.Compare with existing method, method of the present invention has validity for the UGT1A gene of analyzing high beauty on time and cost.

In operation of the present invention (a), described biological specimen picks up from the mankind, preferably comprises high beauty, Chinese and Japanese's Aisa people, and more preferably high beauty.Described biological specimen can comprise blood, skin cells, myxocyte or hair, more preferably blood.

In operation of the present invention (b), the biological specimen that described nucleic acid extraction is collected in operation (a).Described nucleic acid can comprise DNA or RNA, preferably DNA, more preferably genomic dna.The technology of extraction nucleic acid can be carried out according to technology known in the art without limits from collected sample.Alternatively, can use DNA or RNA to extract test kit, for example the test kit of producing by Quiagen company (U.S.) and Stratagene company (U.S.).

In operation of the present invention (c), use nucleic acid of extracting in the operation (b) as template and with primer amplification described UGT1A gene.If the nucleic acid that extracts in the operation (b) is RNA, then this RNA is converted into cDNA to be used as template through reverse transcription.Described primer is designed and makes based on human UGT1A gene or its segmental gene order by currently known methods.

In operation of the present invention (c), preferably increase UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7 and UGT1A9 gene are determined the functional varient in the UGT1A gene.Preferably, amplification UGT1A1, UGT1A6 and UGT1A9 gene determine the UGT1A gene decision to the polymorphism of the susceptibility of Rinotecan.

In operation of the present invention (d), use the UGT1A gene that is increased in the operation (c) to analyze described functional varient or the polymorphism relevant with the drug susceptibility of UGT1A gene.Can use polymorphism analyzing method known in the art to analyze described functional varient or polymorphism.For example, can carry out the combination of SNaPshot analysis, electrophoretic analysis, tetra-sodium order-checking or aforesaid method.

Particularly, if the varient of UGT1A gene to be analyzed comprises SNP, then preferably SNaPshot analyzes.In SNaPshot analyzed, use can make with SNP site adjacent areas annealed primer and ddNTP carried out the PCR reaction.The described primer that uses during SNaPshot analyzes is by based on the currently known methods design of the SNP of UGT1A gene with make.For example, design and manufacturing primer make that the base in next-door neighbour SNP site is 3 ' end, comprise and the adjacent annealing gene order in described SNP site, and have added the T base at 5 ' end.Preferably, the length of the described gene order of annealed is approximately 20bp.If determine several SNP simultaneously, the length of the T base of then described SNaPshot primer 5 ' end is designed to difference, thereby changes the length of described PCR product.

Contain the SNaPshot analysis that primer with reference to 2905～314 gene order can be used for determining the functional varient of UGT1A gene.Contain primer with reference to 315～322 gene order and can be used for determining the UGT1A gene and relevant to the susceptibility of Rinotecan polymorphism.

Can be with known sequence measurement analysis by the gene order of the analysing amplified PCR product of described SNaPshot.Preferably, the gene order of described PCR product can be analyzed with the automatic sequencing method, but is not limited thereto.

If UGT1A gene variant to be analyzed is not SNP (for example ,-39 (TA) 6 in the UGT1A1 gene〉(TA) 7), then can carries out known tetra-sodium and check order and replace described SNaPshot to analyze.The expression of the PPi (inorganic pyrophosphate) that discharges is estimated in described tetra-sodium order-checking when the DNA polymerization.In an exemplary embodiment of the present invention, can use the primer that contains with reference to 292～294 gene order to carry out the tetra-sodium order-checking to determine-39 (TA) 6 of UGT1A1 gene〉(TA) 7.

Hereinafter will be elaborated to illustrative embodiments of the present invention.Following illustrative embodiments has been carried out exemplary illustration to the present invention, but the present invention is not limited to following illustrative embodiments.

<CYP1A2>

Illustrative embodiments 1: determine high beauty CYP1A2 gene genotype

＜1-1〉amplification of CYP1A2 gene

Behind 48 health objects collection blood, the genomic dna separating kit that uses Qiagen company to produce separates DNA from blood.Described CYP1A2 gene comprises 7 exons (exon), and it is long to be approximately 11kb.Described CYP1A2 gene is divided into 15 fragments carries out PCR.The primer that uses in each PCR is as shown in table 1.A, the T, G and the C that are write in the gene order in this manual are meant VITAMIN B4, thymus pyrimidine, guanine and cytosine(Cyt).

[table 1] be used to increase primer of CYP1A2 gene and gene order thereof

The position of described primer and described PCR product big or small as shown in table 2.The position of Nucleotide according to Cytochrome P450 (CYP) allelotrope NK ( Http:// www.cypalleles.ki.se/cyp1a2.htm) naming method write.

[table 2] primer location and PCR product size

The reaction conditions of PCR fragment correspondence is as shown in table 3.

[table 3] PCR reaction conditions

The PCR product	Reaction conditions
		CYP1A2p7	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2p6	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP1A2p5	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2p4	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP1A2p3	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2p2	94 ℃ 4 minutes, (94 ℃ 30 seconds, 68.5 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP1A2plela	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2plelb	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP1A2e2a	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2e2b	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP1A2e3	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2e4	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP1A2e5	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2e6	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP1A2e7	94 ℃ 4 minutes, (94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes

＜1-2〉order-checking of PCR product

With the automated DNA sequenator to by illustrative embodiments＜1-1 the PCR product that obtains checks order.The primer is as shown in table 4.

[table 4] order-checking primer

Analyzed according to illustrative embodiments＜1-1 with the automated DNA sequenator complete genome sequence of CYP1A2 gene of amplification.With the gene order of itself and wild-type CYP1A2 gene (with reference to 1) relatively after, found 17 SNP altogether.Its result is as shown in table 5.Through confirming that SNP-2603insA is New type of S NP.

[table 5] is found in the CYP1A2 gene variant of Koryo philtrum

SNP	Name	The rs numbering	The amino acid variation body	Frequency (%)
					-3860G>A	*1C			27.08
-3598G>T		2069519		9.38
					-3594T>G		2069520		9.38
-3113G>A		2069521		12.50
					-2847T>C		2069522		11.46
-2808A>C		12592480		1.04
					-2603insA		-		1.04
-2467delT	*1D	-		43.75
					-1708T>C		2069525		6.25
-739T>G	*1E			7.29
					-163C>A	*1F	762551		55.21
1514G>A	*13	-	G299S	1.04
					2159G>A		2472304		14.58
2321G>C		3743484		9.38
					3613T>C		4646427		6.25
5347C>T	*1B	2470890	N516N	15.63
					5521A>G				14.58

Then, the inventor uses the DNA of the object that comprises the genetic variant of finding among the described SNP as template, has carried out PCR and used the methods analyst identical with aforesaid method the gene order of described amplified production with containing the primer of reference 38 with 39.Its target is to determine whether described New type of S NP is arranged in a strand of described CYP1A2 gene, whether has other varient in same strand, and whether described New type of S NP is to be caused by the similar gene that is positioned at this other position of karyomit(e).

As a result, find this SNP be positioned at promotor-the 2603insA place.A chain of described double-stranded DNA is a varient and another chain is wild-type (Fig. 1).

Illustrative embodiments 2: the haplotype of determining the CYP1A2 varient

17 kinds of CYP1A2 gene variants of Fa Xianing may influence the activity of CYP1A2 enzyme according to its combination in an exemplary embodiment of the present invention embodiment.Existing report is corresponding to the varient of the enzymic activity of some haplotype.Therefore, the inventor with the SNPA1yze software analysis of DYNACOM company the haplotype that causes by determined varient in the illustrative embodiments 1.As a result, found novel high beauty's haplotype of in other race, not found, as shown in table 6.

[table 6]

Illustrative embodiments 3: the selection of htSNP and checking

Report described several haplotype, i.e. the combination of the SNP of CYP1A2 gene may influence the activity of CYP1A2 enzyme.The specifying information of the haplotype that can be generated with the minimum mark inspection.Described minimum mark is called htSNP, is that accurate mark haplotype is necessary, and comprises several combinations.With SNPtagger software ( Http:// www.well.ox.ac.uk/～xiayi/haplotype) selected described htSNP combination, that is optimum set of tags.The example of selected htSNP combination is as shown in Fig. 2～6.Selected htSNP combination is one of optimum set of tags, and wherein " 1 " is meant wild-type, and " 2 " are varients and ' V ' is meant selected htSNP.The selection of htSNP combination can be different with the htSNP combination in Fig. 2～6.

Two times of types and genotype are determined in the described combination of analyze finding with Matlab software (7.1 editions, Mai Siwoke softcom limited, the U.S.) and both do not overlap.Analytical results is used for determining described combination.

According to the checking result, can determine two times of types and genotype, both do not overlap.This refers to, and the selected htSNP combination of the present invention is inequality, and determines that genotypic described analysis is absolutely correct.

Illustrative embodiments 4: in the CYP1A2 promotor, search for genetic variant fast

That determines in illustrative embodiments 1 is found among 17 SNP in the CYP1A2 gene among people from Koryo, carries out SNaPshot and analyzes high-speed search to influence 11 promotor SNP of CYP1A2 enzymic activity.Use the DNA of object to carry out PCR, then amplified production is carried out SNaPshot and analyze as template.The promotor of described CYP1A2 gene is approximately 4,000 bases, and it is as shown in table 7 to be used for the primer of PCR.

[table 7] primer title and gene order

The reaction conditions of described PCR product is as shown in table 8.

[table 8] PCR reaction conditions

The PCR product	Reaction conditions
		CYP1A2_promoter	94 ℃ 1 minute, (98 ℃ 10 seconds, 55 ℃ 30 seconds, 68 ℃ 4 minutes) 35 cycles, 72 ℃ 5 minutes

Residue primer and the dNTP with the PCR product reaction of amplification back may not influence described SNaPshot analysis.In order to remove residue primer and dNTP, 5 μ L PCR products are mixed with 2 μ LExoSAP-IT (USB production) with 37 ℃ of reactions 30 minutes, reacted again 15 minutes so that remaining enzyme deactivation in 80 ℃ then.Primer in use product and the table 9 carries out PCR and makes multiple SNaPshot reactant.Described multiple SNaPshot reactant and PCR reaction conditions are as shown in table 10 and table 11.

[table 9] primer title and gene order thereof

The primer title	Gene order (5 ' → 3 ')	Reference
			-163C/A_F(24)	TTTTAAAGGGTGAGCTCTGTGGGC	64
-739T/G_F(20)	GCCTGGGCTAGGTGTAGGGG	65
			-2847T/C_F(32)	TTTTTTTTTTTTGCCTTCAAACATGCTCTGTT	66
-2808A/C_R(36)	TTTTTTTTTTTTTTTTAAAACTGTGGGATCAACCTG	67
			-1708T/C_F(40)	TTTTTTTTTTTTTTTTTTTTAACCATTCAAAAGGAGG TTG	68
-3860G/A_R(44)	TTTTTTTTTTTTTTTTTTTTTTTTGCATGACAATTGCT TGAATC	69
			-3113G/A_F(48)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTCAAGAGGAAT CCAAAGAGAC	70
-2603A7/A8_R2(52)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTT TAAACATTTTTTT	71
			-3594T/G_R(56)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT ATTTTTAATGTTTTCTT	72
-3598G/T_F(60)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT CTGTAATTTAATTTTTTTAA	73
			-2467delT_F(64)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTGAGCCATGATTGTGGCACA	74

[table 10] multiple SNaPshot reactant

[table 11]

The PCR product	Reaction conditions
		The SNaPshot product	(96 ℃ 10 minutes, 50 ℃ 5 seconds, 60 ℃ 30 seconds) 30 cycles

Reaction mixes 2 μ L SAP (USB) after carrying out fully with 5 μ L SNaPshot products, 80 ℃ of reactions 15 minutes, thereby remove [F] ddNTP.Then 0.5 μ L reactant, 9.25 μ LHi-Di methane amides (ABI) and 0.25 μ L GeneScan-LIZ scale standard thing (ABI) are mixed 5 minutes so that its sex change in 95 ℃.Then, (3130XL GeneticAnalyzer ABI) analyzes described mixture with 3130XL genetic analysis instrument.Income analysis result is as shown in Fig. 7～14.

As shown in FIG., show the color and the position at different peaks according to the difference of the varient of described CYP1A2 gene, thereby be easy to recognize wild-type, have the allelic varient of heterozygosis (heterozygosis) and have the allelic varient (isozygotying) that isozygotys.Analytical procedure of the present invention is saved cost and time, and can analyze described CYP1A2 gene variant easily.

<CYP2A6>

Illustrative embodiments 5: determine high beauty 2A6 gene genotype

＜5-1〉amplification of 2A6 gene

Behind 50 health objects collection blood, the genomic dna test kit that uses Qiagen company to produce separates DNA from blood.Described CYP2A6 gene comprises 9 exons, and it is long to be approximately 6.9kb.Described CYP2A6 gene is divided into 7 fragments carries out PCR.The PCR the primer is as shown in table 12.A, the T, G and the C that are write in the gene order in this manual are meant VITAMIN B4, thymus pyrimidine, guanine and cytosine(Cyt).

[table 12] be used to increase primer and gene order thereof of CYP2A6 gene

^*: the primer of from document [Drug Metab.Pharmacokin, 17 (5): SNP18 (482)-SNP23 (487) (2002)], quoting

The position of described primer and described PCR product big or small as shown in table 13.The position of Nucleotide according to Cytochrome P450 (CYP) allelotrope NK ( Http:// www.cypalleles.ki.se/cyp1a2.htm) naming method write.

[table 13] primer location and PCR product size

The reaction conditions of PCR fragment correspondence is as shown in table 14.

[table 14] PCR reaction conditions

The PCR product	Reaction conditions
		CYP2A6_exon1	94 ℃ 5 minutes, (94 ℃ 30 seconds, 63 ℃ 30 seconds, 72 ℃ 65 seconds) 35 cycles, 72 ℃ 5 minutes
CYP2A6_exon2	94 ℃ 4 minutes, (94 ℃ 30 seconds, 63 ℃ 30 seconds, 72 ℃ 50 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP2A6_exon3，4	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP2A6_exon5	94 ℃ 4 minutes, (94 ℃ 30 seconds, 63 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP2A6_exon6	94 ℃ 4 minutes, (94 ℃ 30 seconds, 63 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP2A6_exon7，8	94 ℃ 5 minutes, (94 ℃ 30 seconds, 66 ℃ 30 seconds, 72 ℃ 60 seconds) 35 cycles, 72 ℃ 5 minutes
		CYP2A6_exon9	94 ℃ 4 minutes, (94 ℃ 30 seconds, 66 ℃ 30 seconds, 72 ℃ 60 seconds) 35 cycles, 72 ℃ 5 minutes

＜5-2〉order-checking of PCR product

With the automated DNA sequenator and contain with reference to 76～89 primer by illustrative embodiments＜5-1 the PCR product that obtains checks order.

With the gene order (with reference to 75) of itself and wild-type CYP2A6 gene relatively after, find 27 SNP altogether.There are 2 to be New type of S NP among 27 SNP.3 SNP have been found by the structural analysis of gene elmination.30 SNP are as shown in table 5 altogether.

[table 15] is found in the CYP2A6 gene variant of Koryo philtrum

SNP	Allelotrope	The position	The amino acid variation body	Frequency (%)
					-495A>G#		Promotor		1.19
-48T>G		Promotor		28.57
					13G>A		Exons 1	G5R	1.19
22C>T		Exons	1	L8L						25.00
					51G>A	*1B12	Exons 1	V17V	14.29
144G>A		Exons 1	Q48Q	1.19
					237G>A		Intron		1.19
411C>T		Intron		1.19
					567C>T		Exon	2	R101X	1.19
1620T>C		Intron		84.52
					1836G>T		Intron		15.48
1890G>C		Intron		1.19
					2134A>G		Exon	4	K194E	2.38
3391T>C	*11	Exon 5	S224P	1.19
					3492C>T		Exon 5	R257R	1.19
3570C>G		Intron		1.19
					5336G>A		Intron		1.19
5628C>T		Intron		2.38
					5636A>C		Intron		2.38
6218A>G		Intron		1.19
					6282A>G		Intron		2.38
6293T>C		Intron		2.38
					6354T>C		Intron		30.95
6458A>T#		Exon	9	N438Y						3.57
					6558T>C	*7	Exon 9	471T	20.23
6582G>T	*5	Exon 9	G479V	1.19
					6600G>T	*8	Exon 9	R485L	5.95
5971G>A+	*4	Gene elmination		16.00
					5983T>G+	*4	Gene elmination		16.00
6091C>T+	*4	Gene elmination		16.00

In table 15, "+" mark be found in because of the varient in the gene that combines portion C YP2A6 gene and CYP2A7 gene due to the CYP2A6 gene elmination (referring to Figure 33, the exons 1 of CYP2A6 gene～8 are removed, and exon 9 parts of described CYP2A6 gene have been replaced exon 9 ends of CYP2A7 gene).Under the prerequisite of the described CYP2A6 gene of hypothesis, described SNP is numbered (because the CYP2A6 gene is deleted, so described SNP is not at the CYP2A6 gene) according to gene order with global existence.

In order to use described SNP to determine deleted haplotype, forward primer has been designed in 5 ' site in the homologous genes sequence of CYP2A6 and CYP2A7 gene, and in exon 9, having designed reverse primer, 9 pairs of CYP2A6 genes of described exon have specificity but do not increase the CYP2A7 gene.Therefore, whole C YP2A6 gene or the gene that combines because of the deleted and described CYP2A7 gene of described CYP2A6 gene can be amplified, and described CYP2A7 gene is not amplified.

From amplification PCR products, select to have specific base for CYP2A6 and CYP2A7 gene.On the basis of the translation initiation codon ATG of described CYP2A6 gene, the border of the 6091C/T base of described CYP2A6 gene (with reference to 75) is similar with the border (reference 104) of the 6521T of CYP2A7 gene.Be not only 6091, the 5971G in the CYP2A6 gene order is different with described CYP2A7 gene with 5983T.

" ^*" be meant by the approval of international CYP NK and be allelic varient.For example, ^*11 are meant to contain and compare the 224th amino acids is become the varient of proline(Pro) by Serine haplotype with wild-type.Described international naming method referring to Http:// www.cypalleles.ki.se/cyp2a6.htmIn table, " # " refers to novel varient.

Illustrative embodiments 6: the haplotype of determining the CYP2A6 gene genotype

27 the CYP2A6 genetic variants in the illustrative embodiments 5 and the varient of 3 CYP2A6 delete flags depend on its array mode and may influence the activity of CYP2A6 enzyme.Therefore, the inventor with the SNPAlyze software analysis of DYNACOM company the haplotype of determined varient in the illustrative embodiments 5.Usually, the varient of selecting to have the frequency 5% or 10% or more is predicted the distribution of haplotype, because the low frequency varient is difficult to the assurance statistical significance.Yet, even the varient frequency that causes amino acid to be replaced low still have significantly functional.Therefore, in the present invention, used 6 high frequency varient-48T〉G, 22C〉T, 51G〉A, 1620T〉C, 1836G〉T and 6354T〉C and 8 the varient 13G that cause amino acid to be replaced〉A, 567C〉T, 2134A〉G, 3391T〉C, 6458A〉T, 6558T〉C, 6582G〉T and 6600G〉T determines described haplotype.In analysis, added can the marker gene deletion varient 5971G A, 5983T G and 6091C T.Used 17 varients to determine described haplotype altogether.Therefore, the distribution of 20 of the Koryo philtrum haplotypes as shown in Figure 15.

Illustrative embodiments 7: the selection of htSNP and checking

Report, several haplotypes, promptly the SNP of CYP2A6 gene makes up, and may influence the activity of CYP2A6 enzyme.The specifying information of the haplotype that can be generated with the minimum mark inspection.Described minimum mark is called htSNP, is that accurate mark haplotype is necessary, and comprises several combinations.With SNPtagger software ( Http:// www.well.ox.ac.uk/～xiayi/haplotype) analyzed 20 haplotypes selecting according to illustrative embodiments 6 gene order selecting described htSNP combination, that is optimum set of tags.

As a result, the htSNP combination of choosing is as shown in Figure 16～21.Selected htSNP combination is optimum set of tags, and wherein " 1 " is meant wild-type, and " 2 " are varients and " V " is meant selected htSNP.

If analyze the genotype of having determined described varient by described htSNP, then its haplotype can be predicted according to analytical results.Yet the combination of different haplotypes may have identical genotype.Determine two times of types and genotype with the described combination that Matlab software (7.1 editions, Mai Siwoke softcom limited, the U.S.) analysis is found, both do not overlap.

According to the gained result, the htSNP that selects according to present embodiment can determine the mutual haplotype that does not overlap.This means that the htSNP combination of selecting according to the present invention is different, and determines that genotypic described analysis is absolutely correct.

Illustrative embodiments 8: quick search functionality varient in the CYP2A6 gene

In the varient of in illustrative embodiments 5, determining that is found in 27 kinds of CYP2P6 genes among people from Koryo and the varient of 3 kinds of mark CYP2A6 gene elmination, change functional described CYP2A6 gene genotype and can be used for determining gene.Carry out SNaPshot and analyze 10 kinds of functional varients of high-speed search, it is one of high speed genotyping technique of CYP2A6 gene that described SNaPshot analyzes.Described 10 kinds of functional varients comprise that 9 kinds change amino acid or affirmation has functional varient-48T〉G, 13G〉A, 567C〉T, 2134A〉G, 3391T〉C, 6458A〉T, 6558T〉C, 6582G〉T and 6600G〉the varient 6091C of T and marker gene deletion〉T.In the middle of the htSNP of Figure 16 combination, the htSNP that chooses comprises that 10 kinds have functional varient.The position of described varient is shown in table 17.

[table 17] made up the position of the varient of selecting by htSNP of the present invention

	Varient	The position
			htSNP 1	SNP 1	-48T>G
htSNP 2	SNP 2	13G>A
			htSNP 3	SNP 5	567C>T
htSNP 4	SNP 8	2134A>G
			htSNP 5	SNP 9	3391T>C
htSNP 6	SNP 11	6458A>T

htSNP 7	SNP 12	6558T>C
			htSNP 8	SNP 13	6582G>T
htSNP 9	SNP 14	6600G>T
			htSNP 10	SNP 15	6091C>T

Particularly, use the DNA of object to carry out PCR, then amplified production is carried out SNaPshot and analyze as template.The primer that is used for PCR is shown in table 18.

The amplification CYP2A6_long primer amplification the whole length of CYP2A6 gene.Therefore, they can not be used for CYP2A6 gene elmination.In order to determine the 6091C of marker gene deletion〉T, should use a pair of primer CYP2A6delF and the CYP2A6delR CYP2A6 that increases ^*4 products.

[table 18] primer title and gene order

The reaction conditions of described PCR product is shown in table 19.

[table 19] PCR reaction conditions

The PCR product	Reaction conditions
		CYP2A6_long	94 ℃ 1 minute, (98 ℃ 20 seconds, 62 ℃ 30 seconds, 72 ℃ 7 minutes 30 seconds) 30 cycles, 72 ℃ 10 minutes
CYP2A6 *4	94 ℃ 5 minutes, (94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute 20 seconds) 35 cycles, 72 ℃ 7 minutes

Residue primer and the dNTP with the amplification PCR products reaction may not influence described SNaPshot analysis.In order to remove residue primer and dNTP, 5 μ L PCR products are mixed with 2 μ LExoSAP-IT (USB production), in 37 ℃ of reactions 30 minutes, reacted again 15 minutes so that remaining enzyme deactivation in 80 ℃ then.Use described product of handling through enzyme and the primer in the table 20 to carry out PCR and make multiple SNaPshot reactant.Described multiple SNaPshot reactant and PCR reaction conditions are as shown in table 21 and table 22.

[table 20] primer title and gene order thereof

The primer title	Gene order (5 ' → 3 ')	Reference
			Mu_-48T>G	GGCTGGGGTGGTTTGCCTTT	92
Mu_13G>A_F	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTC TACCACCATGCTGGCCTCA	93
			Mu_567C>T_R	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTGAAGGTGGCTTGCTCGCCTC	94
Mu_2134A/G_R	TTTTTTGACAGGAACTCTTTGTCCT	95
			Mu_3391T/C_F	TTTTTTTTTTCCCAGCTCTATGAGATGTTC	96

Mu_6458A>T_R	TTTTTTTTTTTTTTTCAGGCCTTCTCCGAAACAGT	97
			Mu_6558T/C_F	TTTTTTTTTTTTTTTTTTTTCTCCCAGTCACCTAAGG ACA	98
Mu_6582G>T_F	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTGT CCCCCAAACACGTGG	99
			Mu_6600G/T_R	TTTTTTTTTTTTTTTTTTTTTTTTTGGAAGCTCATGGT GTAGTTT		100
Mu1ti2A6/7_6091C >T_F	CAGTCATATTTGCAAGTGT	101

[table 21] multiple SNaPshot reactant

(the composition of "+" 1/2 terminal point damping fluid: 200mM Tris-HCl, 5mM MgCl ₂, pH 9; Nucleic Acids Research, 30 (15): 74,2002)

[table 22]

The PCR product	Reaction conditions
		The SNaPshot product	(96 ℃ 10 seconds, 50 ℃ 5 seconds, 60 ℃ 30 seconds) 40 cycles

After reaction is finished, 1 μ L SAP (USB) is mixed with 10 μ L SNaPshot products,,, thereby remove residue ddNTP then in 65 ℃ of reactions 15 minutes in 37 ℃ of reactions 60 minutes.Then 0.5 μ L reactant, 9.3 μ L Hi-Di methane amides (ABI) and 0.2 μ L GeneScan-LIZ scale standard thing (ABI) are mixed 5 minutes so that its sex change in 95 ℃.Then, analyze described mixture with 3130XL genetic analysis instrument (ABI).Income analysis result is as shown in Figure 22～30.Described genotypic analysis is shown in table 23.

[table 23]

As shown in Figure 22～30, the color at peak is identical with size in each SNP.The color at peak and size are different and different with the functional varient of described CYP2A6 gene, thereby are easy to recognize wild-type, have the allelic varient of heterozygosis (heterozygosis) and have the allelic varient (isozygotying) that isozygotys.In the figure of Figure 22～30, X-axis is meant the miles of relative movement of primer in the automated DNA sequenator that causes because of primer length difference, and Y-axis is meant the intensity of fluorescence that fluorescent material sent with specific wavelength that comprises in each base.

Figure 31 and Figure 32 illustrated with Figure 22～30 in the SNaPshot that together carries out of gene studies analyze, thereby the CYP2A6 gene elmination the genetic variant of research in Figure 22～30.Figure 31 has illustrated the CYP2A6 gene that is present in usually in the homologous chromosomes, and Figure 32 has illustrated the CYP2A6 that is not present in the karyomit(e) and has only a gene.

SNaPshot with the exploitation according to the present invention has analyzed 50 samples and full length sequence thereof.According to described analysis, gene type result 100% is identical.That is to say that the method for the invention has high reproducibility and accurate.

Therefore, can determine the functional varient of CYP2A6 gene easily and save time and cost with analytical procedure of the present invention.

Described method has determined that 10 mainly are found in the CYP2A6 haplotype of Koryo philtrum and have determined described CYP2A6 genotype fast with described combination simultaneously.Owing to comprised the genetic variant that is found in the Koryo philtrum, therefore described analytical procedure is determining that aspect the genotype be very accurately.In addition, described method can be analyzed the Japanese nearly all genotype that has with the closely similar genetics characteristic of high beauty.Think also that in addition described method can be used for definite Chinese CYP2A6 genotype more than 90%.

<CYP2D6>

Illustrative embodiments 9: the CYP2D6 gene genotype of determining high beauty

＜9-1〉separation of genomic dna

Use genomic dna separating kit (Giagen) isolation of genomic DNA from the blood sample that picks up from 174 high beauties.

＜9-2〉amplification of CYP2D6 gene and total length order-checking

From illustrative embodiments＜9-1〉in isolating totally 174 genome DNA samples 51 samples of picked at random as template.Use a pair of primer to carry out 9 exon and the 1.8kb promotor of PCR with amplifying human CYP2D6 gene.

[table 24] is used for the primer of gene amplification

The primer title	Gene order (5 ' → 3 ')	Reference
			CYP505	CACTGGCTCCAAGCATGGCAG	106
3′2D6	ACTGAGCCCTGGGAGGTGGTA	107

Described PCR carried out 1 minute at 94 ℃, carried out 10 seconds at 98 ℃, carried out 30 seconds at 64 ℃, and carried out 7 minutes at 72 ℃, so moved for 30 cycles, and finally carried out 10 minutes at 72 ℃.As a result, produced 6, the PCR product (referring to table 25) of 569bp.

[table 25] primer location and PCR product size

Use amplification PCR products as template, with the gene order of the analysing amplified CYP2D6 gene of totally 13 primers in the table 26.

[table 26] is used for the primer of total length order-checking

The primer title	Gene order (5 ' → 3 ')	Reference
			CYP505	CACTGGCTCCAAGCATGGCAG	106
3′2D6	ACTGAGCCCTGGGAGGTAGGTA	107
			CYP507	AACGTTCCCACCAGATTTC	108
CYP509	GTAAGTGCCAGTGACAGATAAG	109
			2d6-11	AGGATCCTTTGTTCAGGATATGTTGC		110
2d6-12	CACCAAGTACCCCACTTCCC	111
			2d6-1	CATGTGGACTTCCAGAACACACC	112
2d6-2	GGTTCAAACCTTTTGCACTG	113
			2d6-3	GTCGTGCTCAATGGGCTG	114
2d6-4	AAGGTGGATGCACAAAGAGT	115
			2d6-5	GACCTAGCTCAGGAGGGACT	116
2d6-6	AGCTGGATGAGCTGCTAACT	117
			2d6-7	CCTGACCTCCTCCAACATAG	118
2d6-8	CACCTAGTCCTCAATGCCAC	119
			2d6-9	GAGTCTTGCAGGGGTATCAC	120

＜9-3〉at each genotypic analysis respectively

For remaining 123 illustrative embodiments＜9-1〉middle isolating genome DNA sample, analyzed the varient that mainly is found among the Aisa people respectively ^*2A, ^*5, ^*2N, ^*10B, ^*14B, ^*18, ^*21B, ^*41A, ^*49, ^*52 Hes ^*60 genotype.

A) CYP2D6 ^*5 analysis

In order to determine described CYP2D6 ^*5 genotype, the primer in the use table 27 carries out PCR.Described PCR carried out 1 minute at 94 ℃, carried out 10 seconds at 98 ℃, carried out 30 seconds at 64 ℃, and carried out 5 minutes at 72 ℃, so moved for 30 cycles, carried out 10 minutes at 72 ℃ then.As a result, for wild-type, the 5.1kb PCR product that has increased and comprised 9 exons.For CYP2D6 ^*5 varients, 3.5kb PCR product has increased.

The size of gene order of [table 27] primer and position and PCR product

B) CYP2D6 ^*The analysis of 2N

In order to determine described CYP2D6 ^*The 2N genotype, the primer in the use table 28 carries out PCR.Described PCR carried out 1 minute at 94 ℃, carried out 10 seconds at 98 ℃, carried out 30 seconds at 64 ℃, and carried out 8 minutes at 72 ℃, so moved for 30 cycles, carried out 10 minutes at 72 ℃ then.As a result, for CYP2D6 ^*The 2N varient has produced 7.8kb PCR product.

The size of gene order of [table 28] primer and position and PCR product

C) CYP2D6 ^*2 Hes ^*41 analysis

Except that-1584C〉the G varient, CYP2D6 ^*2 genotype and CYP2D6 ^*41 genotype comprise identical varient (1235A〉G;-740C〉T;-678G〉A; Gene is converted to CYP2D7 in introne 1; 1661G〉C; 2850C〉T; 4180G〉C).Use the primer in the table 29 to analyze the gene order that in introne 1 gene is converted to the described varient of CYP2D7 with AS-PCR method (Johanson, Mo1ecular Pharmacology, 46:452-459,1994).

Be the gene conversion of CYP2D7 gene if change in the introne 1, then carry out PCR to produce amplified production with containing with reference to 129 primer 9 and the primer 10B that contains with reference to 125.If described CYP2D6 ^*2 genotype and CYP2D6 ^*41 genotype are normal, then only with contain with reference to 129 primer 9 and contain with reference to 130 primer 10 be combined into performing PCR the time just can produce amplified production.Therefore, can change by determine the described gene that is converted to the CYP2D7 gene in the introne 1 with the existence that is combined into the amplified production that performing PCR produces of described primer 9 and described primer 10B.

Described PCR carried out 5 minutes at 94 ℃, carried out 30 seconds at 94 ℃, carried out 30 seconds at 64 ℃, and carried out 30 seconds at 72 ℃, so moved for 35 cycles, carried out 10 minutes at 72 ℃ then.With the sequencing primer in the table 29 right-1584C the G varient carries out tetra-sodium order-checking.Determined-1584G is CYP2D6 ^*2 genotype and-1584C is CYP2D6 ^*41 genotype.

The gene order of [table 29] primer

D) CYP2D6 ^*10B, ^*14, ^*18 Hes ^*49 genotypic analyses

With PCR-RFLP method (Johanson, Molecular Pharmacology, 46:452-459,1994; Wang, Drug Metabolism and Dispososition, 27:385-388,1998; And Geadigk, Pharmacogenetics, 9:669-682,1999) analyzed described CYP2D6 ^*10B, ^*14, ^*18 Hes ^*49 genotype.The primer is as shown in Table 30, and experiment condition is as shown in table 31.

[table 30] is at the gene order and the position of each genotypic primer

[table 31] at each genotypic Restriction Enzyme and RFLP mutually

Genotype	PCR product size (bp)	Restriction Enzyme	The RFLP phase (bp) of wild-type	The RFLP phase (bp) of varient
					*10B	534	HphI	474+60	376+98+60
*14b	486	MspI	279+207	486

*18	645 o r654	MwoI	348+258+39	272+258+85+39
					*49	486	Sau3A I	347+142	286+142+61

E) CYP2D6 ^*21, ^*52 Hes ^*60 genotypic analyses

By PCR-tetra-sodium sequencing analysis described CYP2D6 ^*21, ^*52 Hes ^*60 genotype.The gene order of the primer that described analysis is used is shown in table 32.

[table 32] is at the gene order of each genotypic primer

Based on the gene sequencing of disclosed CYP2D6 gene among the gene pool accession number M33388 illustrative embodiments＜9-2 and＜9-3 in the data that produce.Each allelic frequency is as shown in table 33.

[table 33] is found in the haplotype of the CYP2D6 gene of Koryo philtrum

In the table 33, " Normal " is meant normal state, and " Incr " is meant increase, and " Decr " is meant reduction, and " None " do not have activity.Mark in the bracket is the writing a Chinese character in simplified form of labeled drug that is used for described analysis: b is bufuralol (bufuralol); D is isocaramidine (debrisoquine); Dx is Dextromethorphane Hbr (dextromethorphan); S is sparteine (sparteine).

After from 12 kinds of genotype that mainly are found in the Koryo philtrum, selecting gene, based on Cytochrome P450 (CYP) allelotrope NK ( Http:// www.cypalleles.ki.se/cyp2d6.htm) each genotypic varient as shown in table 34.In table 34, " 1 " is meant wild-type and " 2 " refer to varient.

[table 34]

Illustrative embodiments 10: the selection of htSNP and checking

Determine to be found in the illustrative embodiments 9 12 CYP2D6 genotype of Koryo philtrum, 33 varients of all in the analytical table 34 are at cost and effective inadequately on the time.Therefore, selected the htSNP of mark genetic variant to come to determine effectively described genotype, described htSNP has the details of haplotype.Essential accurately each haplotype of mark of described htSNP, and comprise various combinations.With SNPtagger software ( Http:// www.well.ox.ac.uk/～xiayi/haplotype/) select described htSNP to make up that is optimum set of tags.The example of selected htSNP combination is as shown in Figure 34～39.Selected htSNP combination is optimum set of tags, and wherein " 1 " is meant wild-type and " 2 " are varients, " V " mark the htSNP that chooses.

Analyze selected combination with Matlab software (7.1 editions, Mai Siwoke softcom limited, the U.S.) and determine mutual two times of types that do not overlap and genotype.Then, determine the htSNP combination.

According to described checking result, can determine two times of types and genotype and both are overlapped.In other words, the htSNP combination of selecting according to the present invention is different, and determines that genotypic described analysis is absolutely correct.

Illustrative embodiments 11: SNaPshot analyzes

Selected htSNP makes up and carries out the SNaPshot analysis in the usage example embodiment 10, and it is one of high speed genotyping technique of CYP2D6 gene that SNaPshot analyzes.HtSNP combination among Figure 34 is selected for analysis.The position of the varient that comprises among the htSNP is shown in table 35.For htSNP1～htSNP3, if analyzed a SNP of various varients, then genotype can be determined.And, have 9 bases to be inserted into and to repeat for htSNP9.Therefore, in the lump the gene order of itself and wild type gene can be determined genotype after relatively when what analyzed the 4125th～4133 bit base.

The position that [table 35] htSNP of the present invention makes up selected varient

htSNP	Varient	The position
			htSNP 1	SNP 2，7，19	-1426 C>T
htSNP 2	SNP 3，6，10，27，30，31	3877 G>A
			htSNP 3	SNP 8，9，12，13，14，15，16，17，18，25	2850 C>T
htSNP 4	SNP 20	1611 T>A
			htSNP 5	SNP 22	1758 G>A
htSNP 6	SNP 23	1887insTA
			htSNP
7	SNP 24	2573insC
			htSNP
8	SNP 26	2988G>A
			htSNP 9	SNP 29	4125-4133ins9bp
htSNP 10	SNP 32	Deletion
			htSNP 11	SNP 33	Repeat

With with illustrative embodiments＜9-2 in identical method increase described CYP2D6 gene to produce about 6.7kb product.For determining CYP2D6 ^*5, use contains the primer CYP2D6_3 (5 ' ACCTCTCTGGGCCCTCAGGGA-3 ') of reference 154 and contains the primer 3 ' 2D6 of reference 123 ^*5 amplification CYP-REP-Del.Described PCR carried out 1 minute at 94 ℃, carried out 10 seconds at 98 ℃, carried out 30 seconds at 64 ℃, and carried out 3 minutes at 72 ℃, so moved for 30 cycles, and finally carried out 10 minutes at 72 ℃.As a result, produced 6, the PCR product of 569bp.

Residue primer and the dNTP with the amplification PCR products reaction may not influence described SNaPshot analysis.In order to remove residue primer and dNTP, 5 μ L PCR products are mixed with 2 μ LExoSAP-IT (USB production), 37 ℃ of reactions 30 minutes, reacted again 15 minutes so that remaining enzyme deactivation at 80 ℃ then.The product that to handle through enzyme and 3 μ L templates (mixture of the CYP-REP-DEL of the CYP2D6 gene of 2 μ L 6.7kb and 1 μ L 3.6kb), the multiple pre-reaction mixture of 1 μ LSNaPshot (ABI), 4 μ L, 1/2 terminal point damping fluid (200mM Tris-HCl, 5mMMgCl ₂, pH 9) and merge the mixing of (Pooled) SNaPshot primer to make totally 10 μ L reactants.Then, the PCR of described reactant was carried out 10 seconds at 96 ℃, carried out 5 seconds, carried out 30 seconds, so moved for 40 cycles at 60 ℃ at 50 ℃.The concentration of treatment of used merging SNaPshot primer is shown in table 36.

[table 36] merges the gene order and the concentration of treatment of SNaPshot primer

The primer title	Gene order (5 ' → 3 ')	Concentration (M)	Reference
				2D6-1426R	GCCACCACGTCTAGCTTTTT	0.05	141
2D6+1611R(P30)	TTTTTTTTTTGGGCCCATAGCGCGCCAGGA	0.3	142
				2D6+1758	CGCCTTCGCCAACCACTCC	0.2	143
2D6+2573(P38)	TTTTTTTTTTTTTTTTTGGGACCCAGCCCAGCC CCCCC	0.02	144
				2D6+2850R(P55)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT CAGGTCAGCCACCACTATGC	0.06	145
2D6+2988(P39)	TTTTTTTTTTTTTTTTTTTTAGTGCAGGGGCCG AGGGAG	0.3	146
				2D6+3877(P45)	TTTTTTTTTTTTTTTTTTTTTTTTTCTGGGCATC CAGGAAGTGTT	0.3	147
2D6+4125(P50)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGCT TCTCGGTGCCCACTG	0.04	148
				2D6+1887R(P60)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTAGGGAGGCGATCACGTTGCT	0.2	152
2D6-5R(P65)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTCTCGTCACTGGTCAGGGGTC	0.05	153

After reaction is finished, 10 μ L reactants are mixed with 1 μ L SAP (USB company), 37 ℃ of reactions 1 hour, then 65 ℃ of reactions 15 minutes.Then 0.5 μ L reactant, 0.2 μ LLIZ120 (ABI) and 9.3 μ L Hi-Di methane amides (ABI) are mixed and place 96 orifice plates.After 2 minutes, analyze described sample 95 ℃ of reactions with 3130 genetic analysis instrument (ABI).Income analysis result as shown in Figure 40.

As shown in figure 40, the color at the peak of each SNP all is identical with size.Can clearly identify wild-type and varient.

In order to analyze the CYP2D6 gene redundancy with SNaPshot,, but use the Dup-F_2 that contains with reference to 155 (5 '-CCTCACCACAGGACTGGCCACC-3 ') herein and contain the Dup-R (5 '-CACGTGCAGGGCACCTAGAT-3 ') of reference 156 producing 3.3kb PCR product with same procedure amplification CYP-REP-Dup.For from described PCR product, removing the residue primer, 5 μ L PCR products and 2 μ L ExoSAP-IT (USB) are mixed, 37 ℃ of reactions 30 minutes.Then, described PCR product makes last ExoSAP-IT inactivation 80 ℃ of reactions 15 minutes.To (contain through PCR product and 3 μ L templates, the multiple pre-reaction mixture of 1 μ L SNaPshot, 4 μ L, 1/2 terminal point damping fluid and the SNaPshot primer that enzyme is handled with reference to 157, CYP2D6-5R, 5 '-CTCGTCACTGGTCAGGGGTC-3 ') mix, make the reactant of 10 μ L, to carry out the SNaPshot reaction under the same conditions.Analyze described reactant with 3100 genetic analysis instrument.Analytical results as shown in figure 41.

As shown in FIG., the color at the peak of SNP is identical with size.Can clearly identify wild-type and varient.

By sequence verification comprise 50 samples of wild-type and genetic variant.100% is identical as a result.In other words, method of the present invention has high reproducibility and accurate.

Described method has determined that 12 mainly are found in the CYP2D6 haplotype of Koryo philtrum and have determined the CYP2D6 genotype fast with described combination simultaneously.Owing to comprised the genetic variant that is found in the Koryo philtrum, therefore described method is determining that aspect the genotype be very accurately.Described method can be used to determine to have the Japanese genotype with the closely similar genetics characteristic of high beauty.Can think that according to The above results described method can be used for definite Chinese CYP2D6 genotype more than 90%.

Illustrative embodiments 12: determine genotype with gene chip

＜12-1〉making of Zip coding chip

1) making of probe

With described probe design is to have and the Zip coding complementary gene order that is used for ASPE PCR reaction.3 ' end insert 10bp nucleotide sequence (5 '-CAG GCC AAGT-3 ') as transcribed spacer to induce the hybridization with target.

5 ' end at described transcribed spacer adds the 24bpZip oligonucleotides coding.(cZip Code) is shown in table 37 for the gene order of described probe.Here, the bold-faced letter in the table 37 is a 10bp transcribed spacer sequence (Figure 43).

[table 37]

The primer title	Gene order (5 ' → 3 ')	Reference
			cZip2	CAGGCCAAGTATCTTGCGCGGCAGCTCGTCGACCG	158
cZip7	CAGGCCAAGTGTGGTCCATCACAAACAGGGAGTCG	159
			cZip8	CAGGCCAAGTCTTGAGCGATGACGGACGGGAAAAG	160
cZip9	CAGGCCAAGTAAGTTGGGGATCTGTAGACCCAGCC	161
			cZip14	CAGGCCAAGTGGATTGCACCGTCAGCACCACCGAG	162
cZip15	CAGGCCAAGTTCCCAGGACGGCGCTGGCACGTTGA
			163
cZip16	CAGGCCAAGTCGGCGTCCACGTCGAGTTCCTTCGC			164
		cZip19	CAGGCCAAGTTTCGGGGAAACTCCGCACCGCCACG		165
cZip20	CAGGCCAAGTTAGGTTTGCCAGTGCGTTGGATCG			166
		cZip21	CAGGCCAAGTTCGACAACCCGGTTGGAGGATTCAG		167
cZip22	CAGGCCAAGTCCAAAAGCTTTACGCCAGCGCCGAA			168
		cZip24	CAGGCCAAGTAGATCGGTGAGCAGTTCAAAGCCGG		169
cZip27	CAGGCCAAGTGGGTATCCGTTCGGTGTTGCGTAGT			170
		cZip31	CAGGCCAAGTTGGTGCTGGCGCAGACCTTTGTCTC		171
cZip32	CAGGCCAAGTACCGCGCAAATGGACAGTGTGGCCA			172
		cZip33	CAGGCCAAGTGACCCCAACTTGACACGTCGCAAGG		173
cZip40	CAGGCCAAGTCGTAAGCCTCGTCAGCTATCCGGGG			174
		cZip41	CAGGCCAAGTCCAAACGCACCCCAACCTGTCCGGA		175
cZip44	CAGGCCAAGTCGGCGGTGGCATTGTCACTGCTGCT			176
		cZip50	CAGGCCAAGTGCAGTTCGTGGCCATGGTGACCGCT		177
cZip56	CAGGCCAAGTCGTTGTGGTAGCGGCACTGGTGGTG			178
		cZip61	CAGGCCAAGTCTGGGTGTGGGTGCTCGTACGCCGA		179
cZip101	CAGGCCAAGTCGGCACATAGGACGGGGTTCAGATA			180
		cZip102	CAGGCCAAGTGAACAAGATTGGTCCTGGAGGTGCG		181
cZip104	CAGGCCAAGTTCGGATGGCGTTCAGTAGGAGAAGG			182
		cZip106	CAGGCCAAGTACACTCTCCATGCGGTAGACCTGAC		183
cZip109	CAGGCCAAGTGAACCTAATGAAGACGGGGGGTGCT
			184

2) point sample of probe and fixing

Use is coated with the Corning product GAPSII slide glass of amine and makes chip.Use OmniGrid100 sample applicator SMP4XP pin point sample on described slide glass.The point sample condition is 22 ℃ and 54% humidity.On 27 probes, carry out the two point sample respectively.After the point sample process finishes, to described slide glass irradiation 7,500 μ L/cm ²UV-light with fixing described probe.

3) be used for the gene chip of Zip encoded test

Use 9 genotype labels (SNP, in table 37 with the bold-faced letter mark) of CYP2D6 gene to verify 11 genotype CYP2D6 ^*1, ^*2, ^*5, ^*10B, ^*14A, ^*14B, ^*18, ^*21, ^*41, ^*49 Hes ^*2N.

[table 38]

CYP2D6 allelotrope	Base changes
		*1	N/A(wt)
*2	1584C>G；1235A>G；2850C>T；4180G>C
		*5	CYP2D6 is deleted
* 10B	100C>T；4180G>C
		*14A	100C>T；1758G>A；2850C>T；4180G>C
*14B	1758G>A；2850C>T；4180G>C
		*18	4125-4133insGTGCCCACT
*21	1584C>G；1235A>G；2573insC；2850C>T
		*41	1584C；1235A>G；2850C>T；4180G>C
*49	1235A>G；100C>T；1611T>A；4180G>C
		*2N	CYP2D6 repeats

＜12-2〉making of target

1) long PCR

With 2 μ L CYP2D6 genome DNA samples, 1X LA damping fluid, 2.5mM MgCl ₂, the primer among the 0.4mM dNTP, 0.2pmol/ μ L table 16, the LA label dna polysaccharase (TAKARA:cat.No.PR002A) and the deionized water of 2.5 units be mixed and made into 50 μ L.Make described mixture carry out 1 sex change 1 minute then,,, 72 ℃ of reactions 6 minutes, and repeat 30 cycles, and then reacted 1 minute so that increase (Figure 44) 64 ℃ of reactions 30 seconds then 98 ℃ of reactions 10 seconds at 94 ℃.(3 kinds of correspondences have been produced from the CYP2D6 gene ^*5, ^*2N allelotrope and other allelic PCR product.Reaction conditions is the same.)

The gene order of [table 39] long PCR primer

2) multiplex PCR

Described long PCR product, 1X AmpliTaq damping fluid, each primer of 0.2mMdNTP, 0.5pmol/ μ L and the AmpliTaq Gold (AppliedBiosystems:cat.No.N8080242) and the deionized water of 0.5 unit that 0.5 μ L is generated are mixed and made into 10 μ L.Make described mixture carry out 1 sex change 5 minutes at 94 ℃, then 94 ℃ of reactions 45 seconds, 57 ℃ 45 seconds, 72 ℃ of reactions 1 minute and repeat 30 cycles, reacted again 1 minute so that increase at 72 ℃ then.The 2nd PCR comprises multiplex PCR.Described PCR product amplification is 4 groups, and is shown in table 40.The gene order of primer is shown in table 41.

[table 40] multiplex PCR group

The gene order of [table 41] multiple PCR primer (genome PCR primer)

3) ASPE (allele-specific primers extension) reaction

Each ASPE primer, the 1 AmpliTaq Gold of unit (Applied Biosystems:cat.No.N8080242), lX Band Doctor (Solgent) and the deionized water of the described multiple PCR products that 6 μ L are generated, 1X AmpliTaq damping fluid, 10 μ M Cy5dUTP (GeneChem), 125nM are mixed and made into 20 μ L.Make described mixture carry out 1 sex change 5 minutes,,,, and repeat 30 cycles increase (referring to Figure 45) 72 ℃ of reactions 1 minute 60 ℃ of reactions 1 minute then 94 ℃ of reactions 30 seconds at 94 ℃.The gene order of described ASPE reaction group and ASPE primer is shown in table 42 and table 43.

[table 42] ASPE reaction group

The gene order of [table 43] ASPE primer

4) PCR purifying

1～4 group of PCR product that the ASPE reaction is generated merges, and carries out purifying with Qiagene purification kit (Qiagen:ca.no.28106) according to manufacturers's handbook.Final efflux volume is 50 μ L.

Using fast vacuum thickner (the 4080C type is made by BioTron) is 1～2 μ L with the PCR product drying behind the purifying.

5) prehybridization of chip

At 42 ℃ of heating prehybridization damping fluids (25% methane amide, 5X SSC, 0.1% SDS and 10mg/mL BSA).Then, chip is immersed in the described damping fluid, and at 42 ℃ of incubations more than 30 minutes.Then described chip is cleaned 3 times with distilled water, put into Taper Pipe, and with separating centrifuge drying 5 minutes under 800rpm.

Then, at 42 ℃ of preheating prehybridization damping fluids (25% methane amide, 5X SSC, 0.1% SDS, 0.5mg/mL poly A, 25 μ g/mL Cot-1DNA, 10% dextran sulfate).With dried sample dissolution in described prehybridization damping fluid.Sample dissolution is put into 0.5mL PCR pipe, 95 ℃ of heating 5 minutes.In hybridization chamber, put into a slice 3M paper, drip 20 μ L3X SSC then thereon.Be loaded into described sample described on the chip of prehybridization, described chipset installed in the hybridization chamber and 42 ℃ of hybridization spend the night then through heating.

With the 0.1% SDS solution that is preheating to 50 ℃ 2X SSC described chip is cleaned 1 time then, lasts 10 minutes, thereafter in room temperature clean 4 times each 1 minute.The chip that cleaned put into canalis spinalis immediately and with separating centrifuge dry 5 minutes at 800rpm.

6) analyze

The output wavelength made from Axon is the described chip that the GenePix 4100B scanner scanning of about 650nm prepares.Fluorescence signal intensity with GenePix Pro 6.0 software analysis scanning images.Described analytical results is as shown in Figure 46 and table 44.

[table 44]

As a result, the varient of the CYP2D6 gene of analyzing with described gene chip is with identical by the varient of sequencing analysis.

<PXR>

Illustrative embodiments 13: determine high beauty PXR gene genotype

＜13-1〉amplification of PXR gene

Behind 54 health objects collection blood, the genomic dna separating kit that uses Qiagen company to make is isolated DNA from blood.Analyzed the complete genome sequence of PXR gene with ABI 3130XL genetic analysis instrument.As a result, 6 kinds in 18 kinds of functional varients having found up to the present to report.Described PXR gene comprises 9 exons, and it is long to be approximately 38kb.With the exon that contains functional varient is the center, and described PXR gene is divided into 10 fragments, so that described fragment is carried out PCR.The PCR the primer is shown in table 45.A, the T, G and the C that are write in the gene order in this manual are meant VITAMIN B4, thymus pyrimidine, guanine and cytosine(Cyt).

The primer and the gene order thereof of [table 45] amplification PXR gene

The size of the position of described primer and PCR product is as shown in table 46.Write according to the naming method of document [HUMAN MUTATION 11:1.3 (1998)] position of Nucleotide.

The size of position of [table 46] primer and PCR product

The reaction conditions of each PCR fragment correspondence is as shown in table 47.

[table 47] PCR reaction conditions

The PCR product	Reaction conditions
		PXR_5′UTR.1	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 35 seconds) 35 cycles, 72 ℃ 5 minutes
PXR_5′UTR.2	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 35 seconds) 35 cycles, 72 ℃ 5 minutes
		PXR_exon1	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 35 seconds) 35 cycles, 72 ℃ 5 minutes
PXR_exon2	94 ℃ 4 minutes, (94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 30 seconds) 35 cycles, 72 ℃ 5 minutes
		PXR_exon3	94 ℃ 4 minutes, (94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 30 seconds) 35 cycles, 72 ℃ 5 minutes

PXR_exon4	94 ℃ 4 minutes, (94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 30 seconds) 40 cycles, 72 ℃ 5 minutes
		PXR_exon5	94 ℃ 4 minutes, (94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 30 seconds) 35 cycles, 72 ℃ 5 minutes
PXR_exon6～8	94 ℃ 4 minutes, (94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 1 minute 15 seconds) 35 cycles, 72 ℃ 5 minutes
		PXR_exon9	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 35 seconds) 35 cycles, 72 ℃ 5 minutes
PXR_exon9.2	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes

＜13-2〉order-checking of PCR product

With the automated DNA sequenator and contain with reference to 131～150 primer analyzed illustrative embodiments＜13-1 the gene order of each PCR product of producing.

With the gene order of wild-type PXR gene (with reference to 130) relatively after, found 22 SNP altogether.Wherein, in 18 kinds of functional varients that have 6 SNP to be contained in to have reported so far.22 SNP have been shown in the table 48, the functional varient # mark of reporting.

[table 48] is found in the PXR gene variant of Koryo philtrum

SNP	The position	The rs numbering	Frequency (%)
				-25564G>A	The upstream	rs12721602	1.9
#-25385C>T	The upstream	rs3814055	16.7
				-24840A>G	The upstream		0.9
-24622A>T	The upstream		2.8
				-24446C>A	The upstream	rs2276705	2.8
-24381A>C	The upstream		21.3
				#-24113G>A	5′UTR		21.3
120A>G	Intron	2						31.5
				155A>G	Intron	2			31.5
178A>T	Intron	2						0.9
				2883T>G	Introne	3	rs3732356		3.7
4500G>A	Intron 4		0.9
				4760G>A	Intron 4	rs3732357	67.6
#7635A>G	Intron	5	rs6785049					51.9
				7675C>T	Intron	5	rs6797879		7.4
7958C>G	Intron	6						0.9
				#8055C>T	Intron	6	rs2276707		37.0
8635C>A	Intron 8		10.2
				9976G>A	3′UTR	rs3732358	0.9
10719A>G	3′UTR		5.6
				#11156A>C	3′UTR		48.1
#11193T>C	3′UTR		48.1

As shown in table 48,7 kinds of varients of described PXR gene are found in the promotor, and all the other varients are found in 3 ' UTR and the intron.There is not to find to cause the varient of amino acid replacement.

Illustrative embodiments 14: the haplotype of determining the functional varient of PXR

Studied 6 kinds of functional varients functional of PXR gene in illustrative embodiments 13, described functional varient may influence the functional of PXR gene because of its array mode.

Therefore, the inventor with the SNPAlyze software analysis of DYNACOM company the haplotype of the varient found in the illustrative embodiments 13.As a result, confirmed that at least 14 kinds have 1% haplotype with upper frequency, shown in table 49.

[table 49]

: the varient of each SNP

Illustrative embodiments 15: the selection of htSNP and checking

Report several haplotypes, the combination of the SNP of promptly described PXR gene may influence the activity of PXR gene.Can check the specifying information of the haplotype that generates with minimum mark.Described minimum mark is called htSNP, is that accurate mark haplotype is necessary, and comprises several combinations.With SNPtagger software ( Http:// www.well.ox.ac.uk/～xiayi/haplotype) to exemplary enforcement side Formula 14In 14 haplotypes selecting check order selecting described htSNP combination that is optimum set of tags.

As a result, the htSNP combination of choosing as shown in Figure 47.Selected htSNP combination is one of optimum set of tags, and wherein " 1 " is meant wild-type, and " 2 " are varients and " V " is meant selected htSNP.The selection of htSNP combination can be different with the htSNP combination among Figure 47.

Mutual two times of types that do not overlap and genotype are determined in the described combination of analyze finding with Matlab software (7.1 editions, Mai Siwoke softcom limited, the U.S.).Analytical results is used for determining described combination.

According to described checking result, two times of types and genotype need not overlap and just can be determined.In other words, the htSNP combination of selecting according to the present invention is different, and determines that genotypic described analysis is absolutely correct.

Illustrative embodiments 16: quick search functionality varient in the PXR gene

In 6 kinds of functional varients of the high beauty PXR gene of in illustrative embodiments 13, finding, carry out SNaPshot and analyze high-speed search to influence the functional varient of described PXR gene function.Use the DNA of object to carry out PCR, amplified production is carried out SNaPshot analyze as template.The used primer of described PCR is shown in table 50.

[table 50]

The reaction conditions of described PCR product correspondence is shown in table 51.

[table 51] PCR reaction conditions

The PCR product	Reaction conditions
		PXR_5′UTR.1	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 35 seconds) 35 cycles, 72 ℃ 5 minutes
PXR_exon1	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 35 seconds) 35 cycles, 72 ℃ 5 minutes
		PXR_exon6	94 ℃ 4 minutes, (94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 30 seconds) 35 cycles, 72 ℃ 5 minutes
PXR_exon9.2	94 ℃ 4 minutes, (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes

Balanced mix described 4 through amplification PCR products.May not influence SNaPshot with residue primer that mixes the reaction of PCR product and dNTP analyzes.In order to remove residue primer and dNTP, 5 μ L PCR products are mixed with 2 μ L ExoSAP-IT (USB manufacturing), 37 ℃ of reactions 30 minutes, reacted again 15 minutes so that remaining enzyme deactivation at 80 ℃ then.Use described product of handling through enzyme and the primer in the table 52 to carry out PCR and make multiple SNaPshot reactant.Described multiple SNaPshot reactant and PCR reaction conditions are as shown in table 53 and table 54.

[table 52] primer and gene order thereof

The primer title	Gene order (5 ' → 3 ')	Reference
			25385C>T_F(48)	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGCAATC CCAGGTT	242
24113G>A_F(44)	TTTTTTTTTTTTTTTTTTTTTTTTGTCTCCTCATTTCTAG	243

	GGTG
			7635A>G_F(28)	TTTTTTTTCCATCCTCCCTCTTCCTCTC	244
8055C>T_F(32)	TTTTTTTTTTTTCTGAGAAGCTGCCCCTCCAT	245
			11156A>C_F(36)	TTTTTTTTTTTTTTTTTATAAGGCATTCCACACCTA	246
11193T>C_R(40)	TTTTTTTTTTTTTTTTTTTTATTCCTTTTGCCTTGATTTG	157

[table 53] multiple SNaPshot reactant

[table 54]

The PCR product	Reaction conditions
		The SNaPshot product	96 ℃ 10 seconds, 50 ℃ 5 seconds, 60 ℃ 30 seconds) 40 cycles

After reaction is finished, 10 μ L SNaPshot products and 1 μ L SAP (USB) are mixed,,, thereby remove [F] ddNTP then 65 ℃ of reactions 15 minutes 37 ℃ of reactions 1 hour.Then, 0.5 μ L reactant, 9.3 μ L Hi-Di methane amides (ABI) and 0.2 μ L GeneScan-LIZ scale standard thing (ABI) are mixed 5 minutes so that its sex change at 95 ℃.Then, analyze described mixture with 3130XL genetic analysis instrument (ABI).Analytical results is shown in Figure 48～50.

Shown in Figure 48～50, the color at peak and position are different and different with the functional varient of described PXR gene, thereby can discern wild-type easily, contain the allelic varient of heterozygosis (heterozygosis) and contain the allelic varient (isozygotying) that isozygotys.Therefore, analytical procedure of the present invention can be used for analyzing the functional varient of PXR gene, and saves cost and time.

<UGT1A>

Illustrative embodiments 17: the selection of the genetic variant in the high beauty UGT1A gene

The separation of step 1) genomic dna

After gathering blood from 50 high beauties, use genomic dna separating kit (Qiagen) from blood sample, to isolate DNA.

Step 2) amplification of UGT1A gene and total length order-checking

Isolated 50 genomic dna samples in the step 1 are used as template.A pair of primer in the use table 55 carries out the PCR described UGT1A gene that increases.The gene order of the gene Name ﹠ Location of the UGT1A gene of amplification, the primer title, primer, the size of primer and PCR reaction conditions are shown in table 55.

[table 55]

The analysis of the varient of step 3) UGT1A gene

Analyzed the full length sequence of the described UGT1A gene of amplification in the step 2 with known 3130X genetic analysis instrument (Applied Biosystems).(the gene pool accession number: gene order NT_005120) compares with analytical results and wild-type UGT1A gene.The result is as shown in table 56 and table 57.

[table 56]

[table 57]

Step 17-1) selection of functional varient in the UGT1A gene

Select it is reported the varient relevant based on the polymorphism of the UGT1A gene that is found in 50 Koryo philtrums in the step 3 with improving or reduce enzymic activity.Selected varient is shown in table 58.Although the G766A varient in the UGT1A9 gene is not determined, it is reported that this varient is the functional varient among the Japanese in step 3.Therefore, comprised the G766A varient in the table 58." truncated protein " is meant the albumen that its translation suspends because of mutant.

[table 58]

Step 17-2) selection of the polymorphism relevant of UGT1A gene with drug susceptibility

Metabolic UGT1A1, the UGT1A6 of known participation resistive connection bowelcancer medicine Rinotecan and the polymorphism of UGT1A9 gene have been selected based on the polymorphism of the UGT1A gene that is found in 50 Koryo philtrums in the step 3, shown in table 59.Although in step 3, do not find the G766A varient in the UGT1A9 gene, it is reported that this varient is Japanese functional varient, thereby it has been added in the table 59.

[table 59]

Illustrative embodiments 18: to the analysis of functional varient in the UGT1A gene and the polymorphism relevant with drug susceptibility

18-1) the analysis of the functional varient of UGT1A gene

The blood that picks up from object is studied analyzing its functional varient, described object contain the UGT1A gene wild-type, contain the allelic varient of heterozygosis and contain the allelic varient that isozygotys.

With with illustrative embodiments 17 in the increased gene order of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7 and UGT1A9 gene of step 1 method identical with step 2.The PCR product of 5 each UGT1A gene of μ L and 2 μ L ExoSAP-IT (USB manufacturing) are mixed be incorporated in 37 ℃ of reactions 30 minutes to remove the residue primer.Then, the reactant that is produced reacted 15 minutes so that remain the ExoSAP-IT inactivation at 80 ℃ again.Then the described reactant of 2 μ L and the 1 multiple pre-reaction mixture of μ L SNaPshot (ABI), 4 μ L, half terminal point damping fluid (are formed: 200mM Tris HCl, 5mM MgCl ₂, pH 9) and table 60 in each SNaPshot primer mix reaction soln with preparation SNaPshot.Herein, the total amount of described reactant is 10 μ L.

[table 60]

Every kind of reaction soln is carried out 40 cycles of PCR (96 ℃ of reactions 10 seconds, 50 ℃ of reactions 5 seconds and 60 ℃ of reactions 30 seconds) under the following conditions.After the reaction, 10 μ L reaction solns and 1 μ L SAP (shrimp alkaline phosphotase) (USB) are mixed and are incorporated in 37 ℃ of reactions 1 hour then 65 ℃ of reactions 15 minutes.0.5 μ L reaction soln mixed with 0.2 μ L LIZ120 (ABI) and 9.3 μ LHi-Di methane amides and place 96 orifice plates.Response sample is analyzed by 3130X genetic analysis instrument (Applied Biosystems) then 95 ℃ of reactions 2 minutes.Analytical results is shown in Figure 51～54.

Shown in Figure 51～54, the color at peak and position are different and different with the functional varient of each UGT1A gene, thereby can identify wild-type easily, contain the allelic varient of heterozygosis (heterozygosis) and contain the allelic varient (isozygotying) that isozygotys.Result 100% by order-checking gained in the illustrative embodiments 17 in the size that has confirmed the peak and type and the table 55 is identical.Therefore, analytical procedure of the present invention can be used for the functional varient of analysis of UG T1A gene, and saves cost and time.

Because the UGT1A1 gene-the 39insTA genotype is not corresponding with SNP, and therefore can't carry out SNaPshot and analyze this genotype.The genotypic varient of described-39insTA is determined by the order-checking of PCR-tetra-sodium.The gene order of primer that is used for described analysis is as shown in table 61.Primer UGT1A1 ^*28F has connected vitamin H (biotin) (referring to reference to 202) at 5 ' end.The primer that is used for the tetra-sodium order-checking is referring to document [ClinChem., Jul; 49 (7): 1182-1185,2003].

More specifically, the PCR product that will generate with the primer that contains reference 202 and 203 is as template.Make with reference to 204 order-checkings with primer reaction, thereby with the existence of tetra-sodium sequenator definitive variation body.

Binding buffer liquid (forming: 10mMTris-HCl, 2M NaCl, 1mM EDTA and 0.1% Tween20) and efficient Streptavidin agarose (the Streptavidin Sepharose of 3 μ L with the PCR product that generated and 37 μ L pH 7.6 ^TMHigh Performance AmershamBioscience) mixes.Then, described mixture is placed 96 orifice plates, in room temperature and 14,000rpm reaction 5 minutes.0.3 μ L is contained the primer (100pmol) of reference 204 (to be formed: 20mM Tutofusin tris acetate and 2mMMgAc with the 1X annealing buffer of 100 μ LpH 7.6 ₂) mix and place 96 orifice plates.Handle described response sample with vacuum Prep Tool, heat 3 minutes cool to room temperature then at 90 ℃.Enzyme mixture, substrate mixture, dATP, dCTP, dGTP and dTTP that Pyro Gold test kit (Biotage) is provided add in the refrigerative plate to pass through tetra-sodium sequenator definitive variation body.

[table 61]

18-2) the analysis of functional varient in the UGT1A gene

Analyzed the UGT1A gene and relevant to the susceptibility of Rinotecan polymorphism, the primer in the primer substitution list 60 in using table 62, method therefor and 18-1) described in SNaPshot analyze identical.Analytical results is shown in Figure 55.5 ' end of the primer in table 62 has added the different T duplicated gene sequence of length changing the length of described primer.

[table 62]

As a result, identify the UGT1A gene and relevant to the susceptibility of Rinotecan several SNP at an easy rate simultaneously.Result 100% in the table 55 that obtains by order-checking in the size that has confirmed the peak and type and the exemplary embodiment 17 is identical.

Industrial applicibility

As mentioned above, the analytical procedure of the functional varient of CYP1A2 of the present invention, CYP2A6, CYP2D6, PXR and UGT1A gene or the polymorphism relevant with drug susceptibility can be used for the polymorphism relevant with drug susceptibility of the functional varient or the UGT1A gene of definite CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene, and described method is used based on the optimum search combination of the polymorphism of still undetermined high beauty CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene before.The present invention can be applied to determine such as to high beauty Japanese and Asian CYP1A2 such as Chinese and high beauty, CYP2A6, CYP2D6, PXR and the UGT1A gene genotype of similar genetics characteristic being arranged.

Although this paper shows illustrative embodiments more of the present invention and illustrates, but one skilled in the art will recognize that, can change described illustrative embodiments under the situation that does not deviate from principle of the present invention and essence, scope of the present invention limits in claims and equivalent.

Claims (80)

1. select the method for the haplotype tagged single-nucleotide polymorphic of human CYP1A2 gene, described method comprises:

(a) collection of biological sample from object;

(b) in the collected described sample of operation (a), extract nucleic acid;

(c) carry out PCR (polymerase chain reaction) with primer, this reaction uses the described nucleic acid that is extracted in the operation (b) to come amplifying human CYP1A2 gene or its fragment as template;

(d) (c) middle gained PCR product checks order and the existence of definitive variation body to operating; With

(e) according to operation determined in (d) to have varient described PCR product gene order prediction haplotype and with SNPtagger software described PCR product is checked order.

2. the method for claim 1, wherein the middle described biological specimen of collecting of operation (a) is selected from blood, skin cells, myxocyte and hair.

3. the described primer of the method for claim 1, wherein operating in (c) is selected from the primer that contains with reference to 2～31.

4. the described varient of the method for claim 1, wherein operating in (d) is selected from single nucleotide polymorphism (SNP), gene elmination and gene redundancy.

5. the method for claim 1, wherein the described order-checking of operation in (d) comprises by automatic sequencing or tetra-sodium and checking order.

6. the method for claim 1, wherein described operation (d) by contrasting described PCR product gene order and the gene order of wild-type CYP1A2 gene carry out.

7. the method for claim 1, described method also comprise and repeat described operation (a)～(d).

8. determine the method for the haplotype of human CYP1A2 gene, described method comprises:

(a) collection of biological sample from object;

(b) extract genomic dna in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described genomic dna that is extracted in the operation (b) to come amplifying human CYP1A2 gene or its fragment as template; With

(d) existence of at least 11 kinds of varients in definite CYP1A2 gene in the gene order of the PCR product of gained in operation (c), described at least 11 kinds of varients are selected from-3860G〉A ,-3598G〉T ,-3594T〉G ,-3113G〉A ,-2847T〉C ,-2808A〉C ,-2603insA ,-2467delT ,-1708T〉C ,-739T〉G ,-163C〉A, 1514G〉A, 2159G〉A, 2321G〉C, 3613T〉C, 5347C〉T and 5521A〉G.

9. method as claimed in claim 8, wherein, the described primer in the operation (c) is selected from the primer that contains with reference to 2～31,62 and 63.

10. method as claimed in claim 8, wherein, described operation (d) comprise determine SNP (single nucleotide polymorphism)-3860G A ,-3598G T ,-3113G A ,-2808A C ,-2603insA ,-2467delT ,-163C A, 1514G A, 2159G A, 5347C T and 5521A the existence of G.

11. method as claimed in claim 8, wherein, described operation (d) comprises definite single nucleotide polymorphism-3860G〉A ,-3113G〉A ,-2808A〉C ,-2603insA ,-2467delT ,-739T〉G ,-163C〉A, 1514G〉A, 2159G〉A, 5347C〉T and 5521A〉existence of G.

12. method as claimed in claim 8, wherein, described operation (d) comprises definite single nucleotide polymorphism-3860G〉A ,-3598G〉T ,-3594T〉G ,-3113G〉A ,-2808A〉C ,-2603insA ,-2467delT ,-163C〉A, 1514G〉A, 2159G〉A, 5347C〉T and 5521A〉existence of G.

13. method as claimed in claim 8, wherein, described operation (d) comprises definite single nucleotide polymorphism-3860G〉A ,-3598G〉T, 2321G〉C ,-3113G〉A ,-2808A〉C ,-2603insA ,-2467delT ,-163C〉A, 1514G〉A, 2159G〉A, 5347C〉T and 5521A〉existence of G.

14. method as claimed in claim 8, wherein, determining to comprise and analyze the existence of determining described varient the existence of described varient in the operation (d) with SNaPshot.

15. method as claimed in claim 14, wherein, described SNaPshot analyzes with being selected from the primer that contains with reference in 64～74 the primer and carries out.

16. detect the method for the varient in the CYP1A2 promoter gene, described method comprises:

(a) collection of biological sample from object;

(b) extract genomic dna in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described genomic dna that is extracted in the operation (b) to come the promoter region of amplifying human CYP1A2 gene as template; With

(d) existence of single nucleotide polymorphism in definite CYP1A2 gene in the gene order of PCR product of gained in operation (c), described single nucleotide polymorphism comprises-3860G〉A ,-3598G T ,-3594T G ,-3113G A ,-2847T C ,-2808A C ,-2603insA ,-2467delT ,-1708T C ,-739T G and-163C A.

17. as claim 8 or 16 described methods, wherein, the described biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

18. method as claimed in claim 16, wherein, the described primer in the operation (b) comprises with reference to 62 and 63.

19. method as claimed in claim 16, wherein, determining to comprise and analyze the existence of determining described varient in operation (d) to the existence of described varient with SNaPshot.

20. method as claimed in claim 19, wherein, described SNaPshot analyzes with being selected from the primer that contains with reference in 64～74 the primer and carries out.

21. select the method for the haplotype tagged single-nucleotide polymorphic in the human CYP2A6 gene, described method comprises:

(a) collection of biological sample from object;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described nucleic acid that is extracted in the operation (b) to come amplifying human CYP2A6 gene or its fragment as template;

(d) existence of definitive variation body in the gene order of the PCR product of gained in operation (c);

(e) determine haplotype by the gene order of the PCR product of having determined to have described varient in operation in (d); With

(f) with SNPtagger software determined described haplotype in the operation (e) is checked order and selects the haplotype tagged single-nucleotide polymorphic.

22. method as claimed in claim 21, wherein, the described biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

23. method as claimed in claim 21, wherein, the described primer in the operation (c) is selected from the primer that contains with reference to 76～89.

24. method as claimed in claim 21, wherein, the described varient in the operation (d) is selected from single nucleotide polymorphism, gene elmination and gene redundancy.

25. method as claimed in claim 21 wherein, determines to comprise the gene order that contrasts described PCR product and the gene order of wild-type CYP2A6 gene to the existence of described varient in the operation (d).

26. method as claimed in claim 21, described method also comprise repetitive operation (a)～(d).

27. determine the method for human CYP2A6 gene genotype, described method comprises:

(a) collection of biological sample from object;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described nucleic acid that is extracted in the operation (b) to come amplifying human CYP2A6 gene or its fragment as template; With

(d) existence of the varient in definite CYP2A6 gene in the gene order of the PCR product of gained in operation (c), described varient comprises :-48T〉G; 13G〉A; 567C〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; 6582G〉T; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G.

28. method as claimed in claim 28, wherein, the described biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

29. method as claimed in claim 27, wherein, the described primer in the operation (c) is selected from the primer that contains with reference to 90,91,102 and 103.

30. method as claimed in claim 27, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises :-48T〉G; 13G〉A; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; 6582G〉T; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G.

31. method as claimed in claim 27, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises :-48T〉G; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 3391T〉C; 6458A〉T; 6558T〉C; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G.

32. method as claimed in claim 27, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises: 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 3391T〉C; 6354T〉C; 6458A〉T; 6558T〉C; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G.

33. method as claimed in claim 27, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises :-48T〉G; 13G〉A; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; Be selected from 6091C T, 5971G A and 5983T a kind of among the G.

34. method as claimed in claim 27, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises :-48T〉G; 13G〉A; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 3391T〉C; 6458A〉T; 6558T〉C; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G.

35. method as claimed in claim 27, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises :-48T〉G; 22C〉T; 51G〉A; 567C〉T; 1620T〉C; 1836G〉T; 2134A〉G; 3391T〉C; 6458A〉T; 6558T〉C; 6600G〉T; Be selected from 6091C T, 5971G A and 5983T a kind of among the G.

36. method as claimed in claim 27, wherein, determining to comprise and analyze the existence of determining described varient the existence of described varient in the operation (d) with SNaPshot.

37. method as claimed in claim 36, wherein, described SNaPshot analyzes with being selected from the primer that contains with reference in 92～101 the primer and carries out.

38. select the method for the haplotype tagged single-nucleotide polymorphic of human CYP2D6 gene, described method comprises:

(a) from human collection of biological sample;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described nucleic acid that is extracted in the operation (b) to come amplifying human CYP2D6 gene or its fragment as template;

(d) existence of definitive variation body in the gene order of the PCR product of gained in operation (c);

(e) determine haplotype by the gene order of the PCR product of having determined to have described varient in operation in (d); With

(f) with SNPtagger software determined described haplotype in the operation (e) is checked order and selects the haplotype tagged single-nucleotide polymorphic.

39. method as claimed in claim 38, wherein, the described biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

40. method as claimed in claim 38, wherein, the described primer in the operation (c) comprises the gene order that is selected from reference to 106,107,121～127,129～136,138,139,149 and 150.

41. method as claimed in claim 38, wherein, the described varient in the operation (d) is selected from single nucleotide polymorphism, gene elmination and gene redundancy.

42. method as claimed in claim 38 wherein, determines to comprise a kind of existence of determining described varient with in order-checking, electrophoretic analysis and the rflp analysis to the existence of described varient in the operation (d).

43. method as claimed in claim 38, described method also comprise repetitive operation (a)～(d).

44. determine the method for human CYP2D6 gene genotype, described method comprises:

(a) from human collection of biological sample;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described nucleic acid that is extracted in the operation (b) to come amplifying human CYP2D6 gene or its fragment as template; With

(d) determine the existence of at least 11 kinds of varients in the CYP2A6 gene, described at least 11 kinds of varients comprise: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from-1028T C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; 1611T〉A; 1758G〉A; 1887insTA; 2573insC; 2988G〉A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

45. method as claimed in claim 44, wherein, the described biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

46. method as claimed in claim 44, wherein, the described primer in the operation (c) comprises the gene order that is selected from the reference 106,107,121～127,129～136,138,139,149 and 150.

47. method as claimed in claim 44, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; 1611T〉A; 1758G〉A; 1887insTA; 2573insC; 2988G〉A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

48. method as claimed in claim 44, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises: be selected from-1584C〉G;-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1611T〉A; 1758G〉A; 2573insC;-740C〉T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; Be selected from-1245insGA ,-1028T〉C ,-377A〉C, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

49. method as claimed in claim 44, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1584C〉G;-1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from-740C T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; 1611T〉A; 1758G〉A; 1887insTA; 2573insC; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

50. method as claimed in claim 44, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises: be selected from-1584C〉G;-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1611T〉A; 1758G〉A; 2573insC;-740C〉T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; Be selected from-1245insGA ,-1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; 4125-4133insGTGCCCACT;-1235A〉G; 1887insTA; The 2D6 deletion; Repeat with 2D6.

51. method as claimed in claim 44, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises: be selected from-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; Be selected from 1611T〉A; 1661G〉C and 4180G〉a kind of among the C; 1758G〉A; 1887insTA; 2573insC; 2988G〉A; 4125-4133insGTGCCCACT;-1235A〉G; 1887insTA; The 2D6 deletion; Repeat with 2D6.

52. method as claimed in claim 44, wherein, described operation (d) comprises the existence of definitive variation body, and described varient comprises: be selected from-1584C〉G;-1426C〉T, 100C〉T and 1039C〉a kind of among the T; Be selected from 1611T〉A; 1758G〉A; 2573insC;-740C〉T ,-678G〉A, 214G〉C, 221C〉A, 223C〉G, 227T〉C, 232G〉C, 233A〉C, 245A〉G and 2850C〉a kind of among the T; Be selected from-1245insGA ,-1028T〉C ,-377A〉G, 3877G〉A, 4388C〉T and 4401C〉a kind of among the T; 1887insTA; 2988G〉A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

53. method as claimed in claim 44, wherein, determining to comprise and analyze the existence of determining described varient the existence of described varient in the operation (d) with SNaPshot.

54. method as claimed in claim 53, wherein, described SNaPshot analyzes and carries out with primer, described primer has the base of the single nucleotide polymorphism of being right after as 3 ' end, have with described mononucleotide polymorphism site adjacent annealed gene order, and have add to 5 ' end the T base.

55. method as claimed in claim 54, wherein, described primer comprises the gene order that is selected from the reference 141～148,152 and 153.

56. determine the method for human CYP2D6 gene with gene chip, described method comprises:

(a) extract gene to be studied and carry out multiplex PCR to obtain to have the PCR product on single nucleotide polymorphism border to be identified;

(b) ASPE (allele-specific primers extension) primer is carried out the ASPE reaction to identify each allelic specificity base;

(c) reactant is mixed mutually with described gene chip; With

(d) analyze described chip.

57. method as claimed in claim 56, wherein, described gene chip comprises the probe of the gene order with reference 158～184.

58.CYP2D6 having, gene type test kit, described test kit be used for the Zip oligonucleotides coding chip that single nucleotide polymorphism detects.

59. select the method for the haplotype tagged single-nucleotide polymorphic of functional varient in the human PXR gene, described method comprises:

(a) from human collection of biological sample;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described nucleic acid that is extracted in the operation (b) to come amplifying human PXR gene or its fragment as template;

(d) existence of definitive variation body in the gene order of the PCR product of gained in operation (c);

(e) determine haplotype by the gene order of the PCR product of having determined to have described varient in operation in (d); With

(f) with SNPtagger software determined described haplotype in the operation (e) is checked order and selects the haplotype tagged single-nucleotide polymorphic.

60. method as claimed in claim 59, wherein, the described biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

61. method as claimed in claim 59, wherein, the described primer in the operation (c) is selected from the primer that contains with reference to 221～240.

62. method as claimed in claim 59, wherein, the described varient in the operation (d) is selected from single nucleotide polymorphism, gene elmination and gene redundancy.

63. method as claimed in claim 59, wherein, described operation (d) by contrasting described PCR product gene order and the gene order of wild-type PXR gene carry out.

64. also comprising, method as claimed in claim 59, described method repeat described operation (a)～(d).

65. determine the method for the functional varient in the PXR gene, described method comprises:

(a) from human collection of biological sample;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) carry out PCR with primer, this reaction uses the described nucleic acid that is extracted in the operation (b) to come amplifying human PXR gene or its fragment as template; With

(d) existence of functional varient in definite PXR gene in the gene order of the PCR product of gained in operation (c), described varient is selected from-25385C〉T ,-24113G〉A, 7635A〉G, 8055C〉T, 11156A〉C and 11193T〉C.

66. as the described method of claim 65, wherein, the described biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

67. as the described method of claim 65, wherein, the primer described in the operation (c) is selected from the primer that contains with reference to 221,222,225,226,235 and 239～241.

68. as the described method of claim 65, wherein, determining to comprise and analyze the existence of determining described functional varient the existence of described functional varient in the operation (d) with SNaPshot.

69. as the described method of claim 68, wherein, described SNaPshot analyzes with being selected from the primer that contains with reference in 242～247 the primer and carries out.

70. determine the method for the functional varient in the UGT1A gene, described method comprises:

(a) from human collection of biological sample;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) use the human UGT1A gene of the described nucleic acid amplification that is extracted in the operation (b); With

(d) the described human UGT1A gene that increases in operation (c) is checked order and definite UGT1A gene in the existence of functional varient, described varient is selected from :-39 (TA) 6 in the UGT1A1 gene〉(TA) 7,211G A, 233C〉T and 686C A; 31T in the UGT1A3 gene〉C, 133C〉T and 140T〉C; 31C in the UGT1A4 gene〉T, 142T〉G and 292C〉T; 19T in the UGT1A6 gene〉G, 541A〉G and 552A〉C; 387T in the UGT1A7 gene〉G, 391C〉A, 392G＜A, 622T〉C and 701T〉C; With in the UGT1A9 gene-118T9 T10,726T G and 766G A.

71. determine the UGT1A gene and method to the relevant polymorphism of the susceptibility of Rinotecan, described method comprises:

(a) from human collection of biological sample;

(b) extract nucleic acid in the collected described sample from operation (a);

(c) use the human UGT1A gene of the described nucleic acid amplification that is extracted in the operation (b); With

(d) the described human UGT1A gene that increases in operation (c) is checked order and definite UGT1A gene in the existence of varient, described varient is selected from: the 211G in the UGT1A1 gene〉A, 233C T and 686C A; 19T in the UGT1A6 gene〉G, 541A〉G and 552A〉C; With in the UGT1A9 gene-118T9 T10,726T G and 766G A.

72. as claim 70 or 71 described methods, wherein, the described mankind in the operation (a) comprise high beauty.

73. as claim 70 or 71 described methods, wherein, the described biological specimen in the operation (a) is selected from blood, skin cells, myxocyte and hair.

74. as claim 70 or 71 described methods, wherein, described nucleic acid comprises DNA or RNA.

75. as the described method of claim 74, wherein, described nucleic acid comprises genomic dna.

76. as the described method of claim 70, wherein, the described UGT1A gene in the operation (c) is selected from UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7 and UGT1A9 gene.

77. as the described method of claim 71, wherein, the described UGT1A gene in the operation (c) is selected from UGT1A1, UGT1A6 and UGT1A9 gene.

78. as claim 70 or 71 described methods, wherein, the described order-checking in the operation (d) is undertaken by the combination of SNaPshot, electrophoresis, tetra-sodium order-checking or aforesaid method.

79. as the described method of claim 70, wherein, the described order-checking of operation in (d) contains SNaPshot with reference to 295～314 primer by use and analyzes or use the tetra-sodium order-checking that contains with reference to 292～294 primer to carry out.

80. as the described method of claim 71, wherein, the described order-checking in the operation (d) contains the SNaPshot analysis of the primer of reference 315～322 and carries out by use.

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