CN101522911B - 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 confirming 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 confirming Cytochrome P450 1A2,2A6 and 2D6, PXR and UDP-glucuronyl transferase 1A gene genotype; More specifically, relate to be used for confirming human CYP1A2, CYP2A6, CYP2D6, NR1I2 (=PXR) with the system of selection of the htSNP of the haplotype of UGT1A gene, confirm the method for said gene genotype and be used for the gene chip of said method.

Background technology

From 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 polymorphum of each gene order in single research population.Haplotype provides more accurately than individual polymorphum and more reliable information about genetic diversity.

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 SUV, lipid acid and VITAMINs (Nelson etc.; Pharmacogenetics 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 is claimed 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 theine, leoponex (clozapine), imipramine (imiparamine) and Proprasylyte (propranolol).In addition; The inner synthetic (like 17 beta estradiols, uroporphyrinogen III) of CYP1A2 catalysis; And the bioactivation of carcinogenic substance (like 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 is claimed 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 is participated 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).Said enzyme is mainly produced by liver, and in such as organs such as lung, large intestine, mammary gland, kidney and uterus, expresses (Drus Metab Dispos., 29 (2): 91-5,2001 on a small quantity; AdvDrug Deliv Rev., 18; 54 (10): 1245-56,2002).

Human cell's cytochrome p 450 2D6 (hereinafter is claimed 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 said genes encoding.

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

Said 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.Said Gene Polymorphisms has partly caused the different activities of said enzyme.As everyone knows, the CYP1A2 gene has represented genetic polymorphism.Up to the present, in promotor, exon and the intron of said 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/cyp 1a2.htm), the genotype of 50 kinds of CYP2A6 ( Http:// www.cypalleles.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 through minimum SNP (SNP) thus definite time of haplotype enhancing accurately and cost effectiveness.

Before various medicines are absorbed by the body and discharge, metabolism will be carried out with transhipment always.Cytochrome P450 (CYP) and drug transporter are participated in metabolism and transhipment.Research to the CYP enzyme of participating 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.Said genetic polymorphism is the principal element of clinical effectiveness, result of treatment and spinoff of the medicine substrate of the said enzyme of influence.Some genetic variants cause deposition and the energy metabolism medicine not of enzyme.Other genetic variant partly reduces enzymic activity.For example, according to the different of said 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 difference between individuals is so big; Activity difference between the individuality still is difficult to directly predicts from genotype, occurs great difference because have the protein expression of drug metabolism enzyme or the drug transporter of low correlation between genotype and the phenotype according to the difference of Externality.For these genes, proteic expression is regulated, but not the existence of genetic variant whether, possibly be the prior factor that causes the difference between individuals of metabolic activity.After the expression of said enzyme was induced, a large amount of generation of these enzymes oneself improved activity.The mechanism of induced expression combines with the promotor of target gene through the outer material that will comprise DR and realizes.The representative instance of said DR is known pregnane X acceptor (PXR) of in the NR1I2 gene, expressing.

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 said 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 said drug metabolism enzyme between the individuality depends on said PXR expression of gene amount difference or activity difference, rather than depends on varient albumen.

In this, individual PXR Gene Polymorphisms or said 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 Oxacyclotetradecane,erythromycin deriv breath test or the body is active.These PXR varients are owing to the expression variation of said target gene causes activity change.Therefore, said PXR varient possibly cause medicine or biomolecules activity difference in vivo, and greatly influences the difference between individuals 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.Said UDP-glucuronyl transferase produces the glucuronic acid couplant that has toxic material such as phenol, ethanol, amine and fatty acid cpds etc.; And be converted into water wetted material to these materials to discharge (Parkinson A through bile or urine from human body; Toxicol Pathol.; 24:48-57,1996).

It is reported that said 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.Said 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 said UGT1A gene family varies with each individual.Known to UGT1A1, UGT1A3～UGT1A10 gene existence several genetic polymorphisms (http://galien.pha.ulaval.ca/alleles/alleles.html).The polymorphum of said UGT1A gene has great difference between the race.The activity that has confirmed enzyme is different because of the difference of polymorphum, and polymorphum is the important factor of decision for the susceptibility of pharmacological agent.UGT1A1*6 relevant with UGT1A1*28 (Monaghan G, Lancet, 347:578-581,1996) with Gilbert syndrome.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.Said haplotype can be analyzed by Arlequin, SNPalyze or other similar software.Analyzing all SNPs (SNP) searches for the heritable variation of every kind of haplotype and knows from experience at cost and effective inadequately on the time.

In order to raise the efficiency, haplotype label SNP (htSNP) can be provided system of selection.Said 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 confirmed 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 through the CYP2D6 genetic variant that mainly is found among the white people is carried out method (Sistonen J etc., the ClinChem.2005 Jul that SNaPshot analyzes to confirm 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 that accurately confirms mainly to be found in the Koryo philtrum in a kind of short period of time, CYP2A6, CYP2D6, NR1I2 (=PXR) with the method for the varient of UGT1A gene.The invention provides to the human CYP1A2 that mainly is found in the Koryo philtrum, CYP2A6, CYP2D6, NR1I2 (=PXR) with 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 that is used to confirm to be found in the Koryo philtrum, CYP2A6, CYP2D6, NR1I2 (=PXR) with the htSNP system of selection of the haplotype of UGT1A gene.

In addition, another aspect of the present invention provide use said htSNP confirm human CYP1A2, CYP2A6, CYP2D6, NR1I2 (=PXR) with 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 confirm the method for human CYP2D6 gene genotype.

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

Above-mentioned and/or others of the present invention and advantage realize that through 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 said 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 is 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 confirmed to have varient in operation in (d) is confirmed haplotype; With with SNPtagger software said 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 through the method for confirming human CYP2A2 gene genotype is provided said 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 PCR product of gained in operation (c), said at least 11 kinds of varients be 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 through the method that detects the varient in the CYP1A2 promoter gene is provided said 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) in operation (c), confirm the existence of CYP1A2 genetic variant in the gene order of PCR product of gained, said 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 through the method for confirming human CYP2A6 gene genotype is provided said 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 PCR product of gained in operation (c), said CYP2A6 genetic variant be 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 through the method for confirming human CYP2D6 gene genotype is provided said 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) confirm the existence of at least 11 kinds of varients in the CYP2D6 gene, said at least 11 kinds of varients comprise: be selected from-1426C＞T, a kind of among 100C＞T and the 1039C＞T; Be selected from-1028T＞C ,-a kind of among 377A＞G, 3877G＞A, 4388C＞T and the 4401C＞T; Be selected from-740C＞T ,-a kind of among 678G＞A, 214G＞C, 221C＞A, 223C＞G, 227T＞C, 232G＞C, 233A＞C, 245A＞G and the 2850C＞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 through the method for confirming the PXR gene genotype is provided said 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) in operation (c), study the existence of the genetic variant of PXR gene in the gene order of PCR product of gained, said 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 through the method that the functional varient of confirming the UGT1A gene is provided said 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 existence of functional varient in the also definite said UGT1A gene of the said gene sequencing of amplification in the operation (c), said functional varient is selected from :-39 in the UGT1A1 gene (TA) 6＞(TA) 7,211G＞A, 233C＞T and 686C＞A; 31T＞C in the UGT1A3 gene, 133C＞T and 140T＞C; 31C＞T in the UGT1A4 gene, 142T＞G and 292C＞T; 19T＞G in the UGT1A6 gene, 541A＞G and 552A＞C; 387T＞G in the UGT1A7 gene, 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 through the method with to the relevant polymorphum of the susceptibility of Rinotecan (irinotecan) of confirming the UGT1A gene is provided said 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 said UGT1A gene of the said human UGT1A gene sequencing of amplification in the operation (c), said varient is selected from: the 211G＞A in the UGT1A1 gene, 233C＞T and 686C＞A; 19T＞G in the UGT1A6 gene, 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 confirm that through providing with gene chip the method for human CYP2D6 gene genotype realizes that said 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) said reaction product is mixed with gene chip; (d) analyze said gene chip.

Above-mentioned and/or others of the present invention and the advantage SNaPshot gene type test kit through being provided for confirming the CYP2D6 gene genotype be used for confirming 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 explained 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 in same karyomit(e), to have repeated at least 2 human CYP2D6 genes.

As stated; 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 polymorphum analyzed, said optimum search is made up based on the polymorphum of also not checked high beauty CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene so far.The present invention go for confirming 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 that combines accompanying drawing, above-mentioned and/or others of the present invention will become obvious and be more readily understood, in said accompanying drawing:

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

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

The result of the varient of CYP1A2 promoter gene has been explained in Fig. 7～14, and said varient is the functional varient (here, the X axle representes to depend on the motion of primer molecule quantity, and the Y axle is represented the height at each peak) of the said CYP1A2 gene of selection according to the present invention,

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

Fig. 8 explained the CYP1A2 promotor that is arranged in heterozygosis (hetero) varient-3860G＞A (CYP1A2*1C) ,-2467delT (CYP1A2*1D) and-163C＞A (CYP1A2*1F) varient; Said heterozygosis varient contains a varient and a wild-type in double-stranded DNA

Fig. 9 explained 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 said varient that isozygotys contains two varients in double-stranded DNA,

Figure 10 explained the CYP1A2 promotor that is arranged in the heterozygosis varient-163C＞A (CYP1A2*1F) and-2808A＞C varient,

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

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

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

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

Figure 15 has explained the kind and the frequency of the CYP2A6 haplotype of philtrum in the Koryo that is used to select the htSNP combination according to the present invention;

Figure 16～21 are the htSNP combination of the clear CYP2A6 gene of selecting according to the present invention for example,

Figure 16 explained and has been used for adding 6 kinds of functional varients and confirming the selection that the htSNP of the haplotype of CYP2A6 gene makes up with 3 kinds of genetic variants with called function property through detect target at genetic variant,

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

Figure 18 has explained the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene; Said CYP2A6 gene comprises 8 kinds of varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants of containing the amino acid replacement

Figure 19 has explained the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene; Said CYP2A6 gene comprises 8 kinds of varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants of containing the amino acid replacement

Figure 20 has explained the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene; Said CYP2A6 gene comprises 8 kinds of varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants of containing the amino acid replacement

Figure 21 has explained the selection of another htSNP combination of the haplotype that is used for definite CYP2A6 gene; Said CYP2A6 gene comprises 8 kinds of varient, 3 kinds of varients with CYP2A6 gene elmination mark and 6 kinds of frequent CYP2A6 genetic variants of containing the amino acid replacement

The result to the SNaPshot analysis of selected htSNP combination and the functional genetic variant combination of CYP2A6 has been explained in Figure 22～30,

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

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

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

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

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

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

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

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

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

Figure 31 and 32 has explained that the SNaPshot that carries out with gene studies described in Figure 22～30 analyzes, and said SNaPshot analyzes and confirmed the CYP2A6 gene elmination the said genetic variant in Figure 22～30 in addition,

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

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

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

The htSNP combination of the CYP2D6 gene of selecting according to the present invention has been explained in Figure 34～39,

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

Figure 42 has explained and has used gene chip to confirm the process of CYP2D6 gene genotype;

Figure 43 has explained the probe on the said gene chip that is used for the CYP2D6 gene;

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

Figure 45 has explained the ASPE reaction process;

Figure 46 has explained and has shown the gene chip according to the analytical results of the varient in 12 pairs of CYP2D6 genes of illustrative embodiments;

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

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

Figure 48 explained PXR gene-25385C＞T ,-24113G＞A, 7635A＞G, 8055C＞T, 11156A＞C and the functional varient of 11193T＞C, said varient is wild-type;

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

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

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

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

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

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

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

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

(a) 211G＞A of UGT1A1 gene, 233C＞T and 686C＞A; 19T＞the G of UGT1A6 gene, 541A＞G and 552A＞C; And the 726T＞G of UGT1A9 gene 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

Hereinafter will be with reference to description of drawings illustrative embodiments of the present invention, and wherein identical Reference numeral is represented identical element, and will avoid duplicate explanation as far as possible.

The present invention can supply through the CYP1A2 gene genotype of the varient analysis of high beauty CYP1A2 gene being confirmed 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 supply to confirm the new haplotype of human CYP1A2 gene.

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

(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) confirmed that to operating in (d) the said PCR product with varient checks order with SNPtagger software.

From the not restriction of method that operation is extracted nucleic acid in the collected sample (a), be well known in the art.As other a kind of selection, can use the extraction test kit to extract said 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 said test kit is RNA, it is for use then to produce cDNA through rt.

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 SNP (SNP).Increase said human CYP1A2 gene or its segmental said 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.

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

Said sequence measurement is restriction not, can be method known in the art.For example, can use automated DNA sequenator or carry out tetra-sodium and check order to confirm gene order.The order-checking of said 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.Said dna sequencing can use from the primer with reference to 32～61 and carry out, but is not limited thereto.Can confirm the existence of varient in the operation (d) through the gene order that compares wild-type CYP1A2 gene.Can use the gene order of said 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.Pharmacokinet 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.Said Haploview software is known in the art.More preferably, can from Http:// www.broad.mit.edu/mpg/haploviewDownload said software.

Method of the present invention can comprise the repetition to operation (a)～(d) in addition.In order to confirm variation phase and haplotype thereof, can detect the genotypic frequency of CYP1A2 and from said 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), with the haplotype data of SNPtagger software analysis CYP1A2 gene with selection htSNP.Said SNPtagger software is known in this area, for example Genehunter, Merlin, Allegro, SNPHAP, htSNP finder (based on PCA).More preferably, said 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 confirms two times of types.After confirming human genotype, said genotype is decoded to confirm 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 said genotype should verify then by the diagnosis decoding of being developed according to the present invention whether it has accurately confirmed said genotype.(Mai Siwoke softcom limited (TheMathWorks, Inc.), the U.S.) analyzes and whether by the genetic analysis result said genotype has been carried out correct decoding, thereby carries out said checking can to use Matlab.

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

In another illustrative embodiments of the present invention; The haplotype of having been confirmed 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.Shown in the instance such as Fig. 2～6 of the htSNP combination of selecting according to the present invention.

The said htSNP combination of selecting according to the present invention can be used for confirming the haplotype of human CYP1A2 gene.Therefore, the invention provides the method for the haplotype of confirming human CYP1A2 gene.Said 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 said CYP1A2 gene in the gene order of PCR product of gained in operation (c), said varient be 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 said extraction nucleic acid in the operation (b) is same as described above.

As stated, the fragment of said human CYP1A2 gene is meant the fragment of the known SNP that comprises said human CYP1A2 gene.Be used for the not restriction of said primer of operation (c), can be selected from reference to 2～31.

The said SNP of research can be selected from the htSNP in Fig. 4～6 in the operation (d).The existence of said SNP can be by the research of following varient: among Fig. 4-3860G＞A ,-3598G＞T ,-3113G＞A ,-2808A＞C ,-2603insA ,-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 said 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 confirming high beauty.

The SNP of the CYP1A2 gene in the gene order of the said PCR product in the operation (d) can use polymorphism analyzing method known in the art to confirm.Preferably, said 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 confirm, more preferably analyzes to confirm with SNaPshot.

Said SNaPshot analysis is meant through carry out PCR with primer reacts to confirm that genotypic method, said primer contain near annealing gene order the SNP site (except that the SNP zone) and ddNTP.The said SNaPshot that uses among the present invention designs and makes with the SNP of currently known methods based on the said 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 said the anneal gene order adjacent with the SNP site is approximately 20bp.If confirm several SNP simultaneously, the length of the T base of then said 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 said PCR product at said 5 ' end.Then, with said SNaPshot primer with combine with each SNP complementary ddNTP.Said compsn varies in size because of the difference of SNP.Therefore, can confirm several SNP simultaneously.

Whether correct for confirming with the gene type result of said SNaPshot analysis gained, carried out the another kind of said genotypic method of confirming.Another kind method is restriction not, preferably includes automated DNA order-checking or tetra-sodium order-checking.

11 SNP that are found in 17 kinds of varients in the said CYP1A2 gene of Koryo philtrum are positioned at promoter region.Said 11 SNP comprise by the present invention confirm first-the 2603insA varient.Therefore, the invention provides the method for confirming varient in the human CYP1A2 promoter gene.Said 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 said reaction; With

(d) in operation (c), confirm the existence of varient in the said CYP1A2 gene in the gene order of PCR product of gained, said 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 said extraction genomic dna in the operation (b) is same as described above.

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

The said SNP of the CYP1A2 gene described in the operation (d) in the gene order of PCR product can use polymorphism analyzing method well known in the art to confirm.Preferably, said SNP can analyze with SNaPshot and confirm.The used SNaPshot of the present invention analyzes and can use based on said 11 SNP of CYP1A2 gene and designed primer is carried 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 said 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 said promoter region that influences the CYP1A2 enzymic activity, by the varient of the said CYP1A2 promoter gene of SNaPshot analyzing and testing.As a result, confirm said method of the present invention varient (referring to Fig. 7～14) in the said CYP1A2 promoter gene of high speed detection exactly.

2.CYP2A6

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

Select the method for htSNP of human CYP2A6 gene following 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) confirm the existence of varient in the gene order of PCR product of gained in operation (c);

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

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

Extracting the not restriction of method of nucleic acid in the said sample of from operation (a), collecting, is the currently known methods in this area.As other a kind of selection, can use the extraction test kit to extract said 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 for use then to produce cDNA through rt.

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 SNP (SNP).Increase said human CYP2A6 gene or its segmental said 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.

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

The method of the existence of definitive variation body can comprise varient detection method known in the art in the operation (d).The gene order of preferably, coming wild-type CYP2A6 gene more known in the art with sequential analysis or electrophoretic analysis is (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.In addition, also available rflp analysis comes the cutting phase of the Restriction Enzyme of more said wild-type CYP2A6 gene.The deletion of said CYP2A6 gene or repetition can be through confirming the electrophoretic analysis of PCR product.Said order-checking can be carried out through automated DNA sequenator or tetra-sodium order-checking.

The said haplotype that operation is confirmed in (e) to contain in the gene order of PCR product of varient can be through confirming such as SNPAlyze, Haplotyper, Arlequin supervisor.

Method of the present invention can comprise the repetition to operation (a)～(d) in addition.In order to confirm variation phase, can detect the genotypic frequency of CYP2A6 and from said 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 said haplotype of confirming in (e) with the SNPtagger software analysis is to select htSNP.Be used to select the said software of htSNP to comprise HapBlock, LDSelect, Haploview, htSNP, TagIT and tagSNPs and SNPtagger.Said 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 confirms two times of types.Can use Matlab (Mai Siwoke softcom limited, the U.S.) to verify said htSNP.

In an illustrative embodiments 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 confirmed the haplotype of 14 SNP among selected 30 SNP, thereby has confirmed to have 19 haplotypes altogether of the frequency more than 1%.Said 14 SNP comprise 8 varient and 6 frequent varients of causing the amino acid replacement and containing functional genetic variant.Be used for confirming that the program of said haplotype is not limited to said SNPAlyze.As other a kind of selection; Can use various softwares known in the art; For example, Haplotyper (http://www.people.fas.harvard.edu/～junliu/Haplo/docMain.htm), Arlequin (htt: //the SNP Analyzer (http://www.istech21.com/) that lgb.unife.ch/arlequin) produces with Istech company.

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

Can use the selected htSNP of the present invention to make up to confirm said human CYP2A6 gene genotype.Therefore, the invention provides the method for confirming said human CYP2A6 gene genotype.Said 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 said CYP2A6 gene in the gene order of PCR product of gained in operation (c), said varient be 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 said extraction nucleic acid in the operation (b) is same as described above.

As stated, the fragment of said human CYP2A6 gene is meant the fragment of the known varient that comprises human CYP2A6 gene, for example, and SNP (SNP).The primer that can be used for operation (c) can comprise the primer with reference to 90,91,120 and 130, but is not limited thereto.

Said varient in the operation (d) can be selected from the htSNP in Figure 16～21.For example, the operation (d) in, said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G; And 13G＞A; 51G＞A; 1620T＞C; And 1836G＞T confirms.Said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G and come confirm.

Shown in figure 18, said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G and come confirm.

Shown in figure 19, said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G and come confirm.

Shown in figure 20, said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G and come confirm.

In addition, shown in figure 21, said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G and come confirm.Preferably, said varient is shown in figure 16 confirms.

In the varient in Figure 16, confirm to have the varient that varient functional or that have a very high potential function property has comprised replacement amino acid or caused gene elmination.Therefore, the functional CYP2A6 varient in the varient among Figure 16 be detected, said amino acid replacement varient or gene elmination varient in the varient among Figure 16 can be studied.Gene elmination almost can't be detected by SNP.When the said varient of search is deleted with marker gene, found 6091C＞T varient.Said 6091C＞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 said 6091C＞T varient can be used as gene elmination mark varient.Be used for confirming 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＞A in the said CYP2A6 gene and 5983T＞G varient can substitute said 6091C＞T varient and come the marker gene deletion.

The said 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 said PCR product in the operation (d) can be used polymorphism analyzing method research 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 said varient.

Said SNaPshot analysis is meant through carry out PCR with primer reacts to confirm that genotypic method, said primer contain near anneal sequence the SNP site (except that the SNP zone) and ddNTP.The said SNaPshot that uses among the present invention uses the currently known methods based on the SNP of the said 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 said 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 said the anneal gene order adjacent with the SNP site is approximately 20bp.If confirm several SNP simultaneously, the length of the T base of 5 ' end of then said 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 said PCR product at said 5 ' end.Then, with said SNaPshot primer with combine with each SNP complementary ddNTP.Said compsn varies in size because of the difference of said SNP.Therefore, can confirm several SNP simultaneously.

Then, amplification can be analyzed with the known sequence measurement in this area with the gene order of the said 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 said 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.Through from the htSNP of Figure 16 combination, selecting 10 kinds of functional or potential function property 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 confirm to be found in the CYP2A6 genotype (referring to Figure 22～32) of Koryo philtrum at high speed simultaneously.

Can comprise with the said CYP2A6 gene genotype that the method for the invention is confirmed-48T＞G, 13G＞A, 567C＞T, 2134A＞G, 3391T＞C, 6458A＞T, 6558T＞C, 6582G＞T, 6600G＞T and 6091C＞T.Shown in corresponding with it each genotype and varient such as Figure 22～32.For example, Figure 22 has explained and has contained-genotype of 48T＞G, 6558T＞C and 2134A＞G varient and 7 wild-types.Figure 23 has explained the genotype that contains 567C＞T and 9 wild-types.Said CYP2A6*4 genotype comprises 2A6 deletion varient.Behind deletion CYP2A6 gene from human chromosomal, can not generate enzyme.If the CYP2A6 gene is deleted, then said gene is shaped as the shape that portion C YP2A6 gene and portion C YP2A7 gene combine.Said deletion specificity varient can be confirmed through studying above-mentioned combination gene.

3.CYP2D6

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

Select the method for htSNP of human CYP2D6 gene following 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) confirm haplotype by the gene order of the PCR product of having confirmed to have varient in operation in (d); With

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

Extracting the not restriction of method of nucleic acid in the said sample of from operation (a), collecting, is the currently known methods in this area.As other a kind of selection, can use the extraction test kit to extract said 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 for use then to produce cDNA through rt.

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 SNP (SNP).Increase said human CYP2D6 gene or its segmental said primer can design based on human CYP2D6 gene or its segmental gene order.For example, said 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.

Said varient in the operation (d) comprises SNP, gene elmination and gene redundancy, but is not limited thereto.For example, said varient can comprise 33 kinds of varients, like 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 confirm said varient with gene sequencing, electrophoretic analysis and rflp analysis.Said gene sequencing can be carried out through automated DNA sequenator or tetra-sodium order-checking.The order-checking of said 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 confirm the existence of the varient in the operation (d) through the gene order that compares wild-type CYP2D6 gene.The gene order of said 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 said wild-type CYP2D6 gene.The deletion of said CYP2D6 gene or repetition can be through confirming the electrophoretic analysis of PCR product.

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

Method of the present invention can comprise the repetition to operation (a)～(d) in addition.In order to confirm variation phase, can detect the genotypic frequency of CYP2D6 and from said 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 confirming in (e) with the SNPtagger software analysis is to select htSNP.Said SNPtagger software is known in this area, for example Genehunter, Merlin, Allegro, SNPHAP, htSNP finder (based on PCA), more preferably, said 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 confirms two times of types.After confirming human genotype, said genotype is decoded to confirm two kinds of haplotype combination by double-stranded karyomit(e).If confirm several SNP simultaneously, then the combination of specific haplotype maybe be identical with the combination of another haplotype.If said genotype should verify then by the diagnosis decoding of being developed according to the present invention whether it has accurately confirmed said genotype.Can use Matlab (Mai Siwoke softcom limited, the U.S.) to analyze whether said genotype has been carried out correct decoding, thereby carry out said checking by the genetic analysis result.

In an illustrative embodiments 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.Shown in the instance such as Figure 34～39 of the htSNP combination of selecting according to the present invention.

The said htSNP combination of selecting according to the present invention can be used for confirming human CYP2D6 gene genotype.Therefore, the invention provides the method for confirming human CYP2D6 gene genotype.Said 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), said at least 11 kinds of varients comprise: be selected from-1426C＞T, a kind of among 100C＞T and the 1039C＞T; Be selected from 1028T＞C ,-a kind of among 377A＞G, 3877G＞A, 4388C＞T and the 4401C＞T; Be selected from-740C＞T ,-a kind of among 678G＞A, 214G＞C, 221C＞A, 223C＞G, 227T＞C, 232G＞C, 233A＞C, 245A＞G and the 2850C＞T; 1611T＞A; 1758G＞A; 1887insTA; 2573insC; 2988G＞A; 4125-4133insGTGCCCACT; The 2D6 deletion; Repeat with 2D6.

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

As stated, the fragment of said human CYP2D6 gene is meant the fragment of the known varient that comprises human CYP2D6 gene, for example, and SNP (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.

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

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

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

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

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

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

Preferably, can confirm the existence of the varient among Figure 34.The said varient of confirming 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 use polymorphism analyzing method known in the art to confirm.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 confirm said varient.If the varient among the said CYP2D6 comprises SNP, then can use SNaPshot to analyze.

Said SNaPshot analysis is meant through carry out PCR with primer reacts to confirm that genotypic method, said 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 said CYP2D6 gene of confirming 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 said SNP site as 3 ' end, and has added the T base at 5 ' end and gets final product.Preferably, the length of said the anneal gene order adjacent with the SNP site is approximately 20bp.If confirm several SNP simultaneously, the length of the T base of then said 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 said PCR product at said 5 ' end.Then, with said SNaPshot primer with combine with each SNP complementary ddNTP.Said compsn varies in size because of the difference of said SNP.Therefore, can confirm several SNP simultaneously.

For example, have and be selected from reference to 141～148 and make up with reference to the primer of 152 and 153 gene order can be used for studying Figure 34 in operation (c) htSNP.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 through the gene order of the analysing amplified PCR product of said SNaPshot.Said 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 said 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 confirm to be found in the CYP2D6 genotype (referring to Figure 40 and 41) of Koryo philtrum at high speed simultaneously.

Can comprise CYP2D6*1A, CYP2D6*2A, CYP2D6*5, CYP2D6*2N, CYP2D6*10B, CYP2D6*14B, CYP2D6*18, CYP2D6*21B, CYP2D6*41A, CYP2D6*49, CYP2D6*52 and CYP2D6*60 with the said CYP2D6 gene genotype that the method for the invention is confirmed.Shown in each genotype and corresponding with it varient such as the table 34.Referring to table 34; For example; Said CYP2D6*1A genotype comprises a wild-type, and said CYP2D6*2A genotype comprises in the gene order of wild-type CYP2D6 gene the varient in SNP1, SNP5, SNP8, SNP9, SNP12～SNP18, SNP21, SNP25 and SNP28 site.Said CYP2D6*5 genotype comprises 2D5 deletion varient.After the CYP2D6 gene is deleted fully, no longer produce enzyme from human chromosomal.Said CYP2D6*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 confirm human CYP2D6 gene genotype.Said method comprises the steps:

(a) extract gene to be studied, said 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) said reaction product is mixed with gene chip mutually; With

(d) analyze said gene chip.

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

Each SNP is generated a pair of primer in operation (b), to carry out the ASPE reaction.The ASPE primer that is generated be 3 ' end comprise the SNP site and with allele-specific bonded gene order.Said 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 the different gene sequence 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, said 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 said ASPE primer to carry out the ASPE reaction, then contain allelic sample and the said primer held corresponding to 3 ' of said primer and react so that the allele-specific extension to take place.Carry out said 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).Said fluorescent material is not limited to Cy5, can use other material, such as Cy3, TAMRA, TexasRed, Cy3.5, Rhodamin 6G, SyBR Green etc.

The complementary bonded oligonucleotide probe (cZip Code, complementary Zip coding) of encoding with said 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 said Zip coding primer extension.

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

Said 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 said reaction product being mixed with said gene chip mutually and analyzing in (d) through the said 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 said CYP2D6 gene with gene chip according to the invention, then its result with come to the same thing with what sequential analysis was verified.Therefore, gene chip of the present invention can the low-cost varient of analyzing range gene.

4.PXR

Method of the present invention uses the htSNP that selects based on the varient in the high beauty PXR gene to confirm 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) confirm 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.

Said not restriction of method of from the sample that operation (a) is collected, extracting nucleic acid is the currently known methods in this area.As other a kind of selection, can use the extraction test kit to extract said 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 for use then to produce cDNA through rt.

The fragment of the said human PXR gene in the operation (c) is meant the fragment of the known varient that comprises human PXR gene, for example, and SNP (SNP).Increase said human PXR gene or its segmental primer can design based on human PXR gene or its segmental gene order, and said 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, said varient can comprise 22 kinds of varients, like 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 confirmed said varient.Said gene sequencing can be carried out through automated DNA sequenator or tetra-sodium order-checking.

Can confirm the existence of varient in the operation (d) through the gene order that compares wild-type PXR gene.Can use the gene order of said 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 said wild-type PXR gene.The deletion of said PXR gene or repetition can be through confirming 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.Said Haploview software is known in the art, and more preferably, can from Http:// www.broad.mit.edu/mpg/haploviewDownload said software.

Method of the present invention can comprise the repetition to operation (a)～(e) in addition.In order to confirm variation phase and haplotype thereof, can detect the genotypic frequency of PXR and from said 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.Said SNPtagger software is known in this area, for example Genehunter, Merlin, Allegro, SNPHAP, htSNP finder (based on PCA), more preferably, said 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 confirms two times of types.After confirming human genotype, said genotype is decoded to confirm two kinds of haplotype combination by double-stranded karyomit(e).If confirm several SNP simultaneously, then the combination of specific haplotype maybe be identical with the combination of another haplotype.If said genotype should verify then by the diagnosis decoding of being developed according to the present invention whether it has accurately confirmed said genotype.Can use Matlab (Mai Siwoke softcom limited, the U.S.) to analyze whether said genotype has been carried out correct decoding, thereby carry out said checking by the genetic analysis result.

In an illustrative embodiments 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 confirmed the haplotype of 6 functional varients among 22 selected SNP, thereby has confirmed 14 haplotypes (referring to table 49) altogether.

In another illustrative embodiments of the present invention, with SNPtagger software said 14 haplotypes are checked order to select htSNP, also can confirm 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 confirm the functional varient of human PXR gene.Therefore, the invention provides the method for the functional varient of confirming human PXR gene.Said 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 PCR product of gained in operation (c), said varient be selected from-25385C＞T ,-24113G＞A, 7635A＞G, 8055C＞T, 11156A＞C and 11193T＞C.

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

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

The said 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 said functional varient.

Can confirm 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 confirm the existence of said varient with SNaPshot with SNaPshot.

Said SNaPshot analysis is meant through carry out PCR with primer reacts to confirm that genotypic method, said 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 said 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 said 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 said the anneal gene order adjacent with the SNP site is approximately 20bp.If confirm several SNP simultaneously, the length of the T base of then said 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 said PCR product at said 5 ' end.Then, with said SNaPshot primer with combine with each SNP complementary ddNTP.Said compsn varies in size because of the difference of said SNP.Therefore, can confirm several SNP simultaneously.

Whether correct in order to confirm the gene type result who uses said SNaPshot to analyze, adopted another kind of methods of genotyping.The not restriction of another kind of methods of genotyping preferably includes automated DNA order-checking or tetra-sodium order-checking.

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 said SNaPshot analysis, and analyzed the gene order of gained PCR product.As a result, confirm that method of the present invention can confirm to be found in the PXR gene function property varient (referring to Figure 48～50) of Koryo philtrum at high speed simultaneously.

The functional varient of the said PXR gene that can confirm with method of the present invention comprises-25385C＞T ,-24113G＞A, 7635A＞G, 8055C＞T, 11156A＞C and 11193T＞C.

5.UGT1A

The method of confirming 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) nucleic acid that is extracted in the use operation (b) is amplifying human UGT1A gene individually; With

(d) the said gene that increases in (c) checks order and the existence of the functional varient of definite UGT1A gene to operating, and said varient is selected from :-39 in the UGT1A1 gene (TA) 6＞(TA) 7,211G＞A, 233C＞T and 686C＞A; 31T＞C in the UGT1A3 gene, 133C＞T and 140T＞C; 31C＞T in the UGT1A4 gene, 142T＞G and 292C＞T; 19T＞G in the UGT1A6 gene, 541A＞G and 552A＞C; 387T＞G in the UGT1A7 gene, 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 polymorphum 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) nucleic acid that is extracted in the use operation (b) is amplifying human UGT1A gene individually; With

(d) the said gene that increases in (c) checks order and the existence of definite UGT1A genetic variant to operating, and said varient is selected from: the 211G＞A in the UGT1A1 gene, 233C＞T and 686C＞A; 19T＞G in the UGT1A6 gene, 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 polymorphum set of tags of selecting based on the polymorphum of the UGT1A gene that mainly is found in high beauty, and has confirmed functional varient or the drug susceptibility in the UGT1A gene.Compare with existing method, method of the present invention has validity as far as the UGT1A gene of analyzing high beauty on time and cost.

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

In operation of the present invention (b), the biological specimen that said nucleic acid extraction is collected in operation (a).Said nucleic acid can comprise DNA or RNA, preferably DNA, more preferably genomic dna.From collected sample, extract the not restriction of technology of nucleic acid, can carry out according to technology known in the art.As other a kind of selection, can use DNA or RNA to extract test kit, the test kit of for example 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 said 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 rt.Said 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), the UGT1A1 that preferably increases, UGT1A3, UGT1A4, UGT1A6, UGT1A7 and UGT1A9 gene are confirmed the functional varient in the UGT1A gene.Preferably, amplification UGT1A1, UGT1A6 and UGT1A9 gene confirm the UGT1A gene decision to the polymorphum 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 said functional varient or the polymorphum relevant with the drug susceptibility of UGT1A gene.Can use polymorphism analyzing method known in the art to analyze said functional varient or polymorphum.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 said 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 said SNP site, and have added the T base at 5 ' end.Preferably, the length of the said gene order of annealed is approximately 20bp.If confirm several SNP simultaneously, the length of the T base of then said SNaPshot primer 5 ' end is designed to difference, thereby changes the length of said PCR product.

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

Gene order by the analysing amplified PCR product of said SNaPshot can be used known sequence measurement analysis.Preferably, the gene order of said 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 in the UGT1A1 gene (TA) 6＞(TA) 7), then can carries out known tetra-sodium and check order and replace said SNaPshot to analyze.The expression of the PPi (inorganic pyrophosphate) that when the DNA polymerization, discharges is estimated in said tetra-sodium order-checking.In an illustrative embodiments of the present invention, can use the primer of the gene order that contains reference 292～294 to carry out the tetra-sodium order-checking to confirm-39 (TA) 6＞(TA) 7 of UGT1A1 gene.

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: confirm high beauty CYP1A2 gene genotype

The amplification of < 1-1>CYP1A2 gene

Behind 48 health objects collection blood, the genomic dna separating kit that uses Qiagen company to produce separates DNA from blood.Said CYP1A2 gene comprises 7 exons (exon), and it is long to be approximately 11kb.Said CYP1A2 gene is divided into 15 fragments carries out PCR.The primer that in each PCR, uses is as shown in table 1.The A, 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 said primer and said 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
		CYP1A2p1e1a	94 ℃ 4 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) 35 cycles, 72 ℃ 5 minutes
CYP1A2p1e1b	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

The order-checking of < 1-2>PCR product

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

Primer is used in [table 4] order-checking

Analyzed complete genome sequence with the automated DNA sequenator according to the CYP1A2 gene of illustrative embodiments < 1-1>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 said SNP as template, has carried out PCR and used the methods analyst identical with aforesaid method the gene order of said amplified production with containing the primer of reference 38 with 39.Its target is to confirm whether said New type of S NP is arranged in a strand of said CYP1A2 gene, in same strand, whether has other varient, and whether said 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 said double-stranded DNA is a varient and another chain is wild-type (Fig. 1).

Illustrative embodiments 2: the haplotype of confirming the CYP1A2 varient

17 kinds of CYP1A2 gene variants in illustrative embodiments of the present invention, finding possibly influence the activity of CYP1A2 enzyme according to its combination.Existing report is corresponding to the varient of the enzymic activity of some haplotype.Therefore, the inventor with the SNPAlyze 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 said several haplotype, i.e. the combination of the SNP of CYP1A2 gene possibly influence the activity of CYP1A2 enzyme.The specifying information of the haplotype that can be generated with minimum mark inspection.Said 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 said htSNP combination, that is optimum set of tags.Shown in the instance such as Fig. 2～6 of selected htSNP combination.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 confirmed in the said 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 confirming said combination.

According to the checking result, can confirm 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 confirms that genotypic said analysis is absolutely correct.

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

Among 17 SNP in the CYP1A2 gene of in illustrative embodiments 1, confirming that is found in the Koryo philtrum, carry out SNaPshot and analyze 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 said 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 said 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 possibly not influence said SNaPshot analysis.In order to remove residue primer and dNTP, 5 μ LPCR 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 processes multiple SNaPshot reactant.Shown in said multiple SNaPshot reactant and PCR reaction conditions such as table 10 and the 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 said mixture with 3130XL genetic analysis appearance.Shown in income analysis result such as Fig. 7～14.

Shown in figure, show the color and the position at different peaks according to the difference of the varient of said 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 according to the invention is saved cost and time, and can analyze said CYP1A2 gene variant easily.

<CYP2A6>

Illustrative embodiments 5: confirm high beauty 2A6 gene genotype

The amplification of < 5-1>2A6 gene

Behind 50 health objects collection blood, the genomic dna test kit that uses Qiagen company to produce separates DNA from blood.Said CYP2A6 gene comprises 9 exons, and it is long to be approximately 6.9kb.Said CYP2A6 gene is divided into 7 fragments carries out PCR.The PCR the primer is as shown in table 12.The A, 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): SNP 18 (482)-SNP23 (487) (2002)], quoting

The position of said primer and said 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

The order-checking of < 5-2>PCR product

With automated DNA sequenator and the primer that contains with reference to 76～89 the PCR product that is obtained by illustrative embodiments < 5-1>is checked 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 through 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	I471T	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 has combined 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 said CYP2A6 gene have been replaced exon 9 ends of CYP2A7 gene).Under the prerequisite of the said CYP2A6 gene of hypothesis, said SNP is numbered (owing to the CYP2A6 gene is deleted, so said SNP is not to be to be directed against the CYP2A6 gene) according to gene order with global existence.

In order to use said SNP to confirm the haplotype of being deleted; 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 said exon have specificity but do not increase the CYP2A7 gene.Therefore, whole C YP2A6 gene or because of said CYP2A6 gene can be increased with the gene that said CYP2A7 gene combines by deletion, and said CYP2A7 gene is not increased.

From amplification PCR products, select to have specific base for CYP2A6 and CYP2A7 gene.On the basis of the translation initiation codon ATG of said CYP2A6 gene, the border of the 6091C/T base of said 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 said CYP2A7 gene with 5983T.

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

Illustrative embodiments 6: the haplotype of confirming 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 possibly 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 cause the varient frequency of amino acid replacement 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 varient 13G＞A, 567C＞T, 2134A＞G, 3391T＞C, 6458A＞T, 6558T＞C, 6582G＞T and 6600G＞T that cause amino acid to be replaced to confirm said haplotype.In analysis, added can the marker gene deletion varient 5971G＞A, 5983T＞G and 6091C＞T.Used 17 varients to confirm said 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 possibly influence the activity of CYP2A6 enzyme.The specifying information of the haplotype that can be generated with minimum mark inspection.Said minimum mark is called htSNP, is that accurate mark haplotype is necessary, and comprises several kinds of combinations.With SNPtagger software ( Http:// www.well.ox.ac.uk/～xiayi/haplotype) analyzed 20 haplotypes selecting according to illustrative embodiments 6 gene order selecting said htSNP combination, that is optimum set of tags.

As a result, the htSNP combination of choosing is 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 confirmed said varient through said htSNP, then its haplotype can be predicted according to analytical results.Yet the combination of different haplotypes possibly have identical genotype.Two times of types and genotype are confirmed in said combination with Matlab software (7.1 editions, Mai Siwoke softcom limited, the U.S.) analysis is found, and both do not overlap.

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

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

In the varient of 27 kinds of CYP2P6 genes that are found in the Koryo philtrum of in illustrative embodiments 5, confirming and the varient of 3 kinds of mark CYP2A6 gene elmination, change functional said CYP2A6 gene genotype and can be used for confirming 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 said SNaPshot analyzes.Said 10 kinds of functional varients comprise that 9 kinds of change amino acid or affirmation have the varient 6091C＞T of functional varient-48T＞G, 13G＞A, 567C＞T, 2134A＞G, 3391T＞C, 6458A＞T, 6558T＞C, 6582G＞T and 6600G＞T and marker gene deletion.In the middle of the htSNP of Figure 16 combination, the htSNP that chooses comprises that 10 kinds have functional varient.The position of said 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
			htSNP1	SNP1	-48T＞G
htSNP2	SNP2
			13G＞A
htSNP3	SNP5				567C＞T
		htSNP4	SNP8
	2134A＞G
		htSNP5	SNP9
	3391T＞C
		htSNP6	SNP11	6458A＞T

htSNP7	SNP12		6558T＞C
				htSNP8	SNP13
	6582G＞T
		htSNP9	SNP14
	6600G＞T
		htSNP10	SNP15
	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 confirm the 6091C＞T of marker gene deletion, should use a pair of primer CYP2A6 delF and the CYP2A6 delR CYP2A6*4 product that increases.

[table 18] primer title and gene order

The reaction conditions of said 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 possibly not influence said 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 said product of handling through enzyme and the primer in the table 20 to carry out PCR and process multiple SNaPshot reactant.Shown in said multiple SNaPshot reactant and PCR reaction conditions such as table 21 and the 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	TTTTTTTTTTTTTTTTTTTTCTCCCAGTCACCTAAGGACA	?98
Mu_6582G＞T_F	TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTGTCCCCCAAACACGTGG	?99
			Mu_6600G/T_R	TTTTTTTTTTTTTTTTTTTTTTTTTGGAAGCTCATGGTGTAGTTT	?100
Multi2A6/7_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

Reaction mixes 1 μ L SAP (USB) after accomplishing with 10 μ L SNaPshot products, in 37 ℃ of reactions 60 minutes, then in 65 ℃ of reactions 15 minutes, thereby remove residue ddNTP.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 said mixture with 3130XL genetic analysis appearance (ABI).Shown in income analysis result such as Figure 22～30.Said genotypic analysis is shown in table 23.

[table 23]

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 said 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, the X axle is meant the miles of relative movement of primer in the automated DNA sequenator that causes because of primer length difference, and the Y axle is meant the intensity of fluorescence that fluorescent material sent with specific wavelength that comprises in each base.

Figure 31 and Figure 32 explained 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 explained the CYP2A6 gene that is present in usually in the homologous chromosomes, and Figure 32 has explained the CYP2A6 that is not present in the karyomit(e) and has only a gene.

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

Therefore, can confirm the functional varient of CYP2A6 gene easily and save time and cost with analytical procedure according to the invention.

Said method has confirmed that 10 mainly are found in the CYP2A6 haplotype of Koryo philtrum and have confirmed said CYP2A6 genotype fast with said combination simultaneously.Owing to comprised the genetic variant that is found in the Koryo philtrum, therefore said analytical procedure is confirming that aspect the genotype be very accurately.In addition, said 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 said method can be used for confirming the Chinese CYP2A6 genotype more than 90%.

<CYP2D6>

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

The separation of < 9-1>genomic dna

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

The amplification of < 9-2>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

Said 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	GACCTAGCTCAG?GAGGGACT	116
2d6-6	AGCTGGATGAGCTGCTAACT	117
			2d6-7	CCTGACCTCCTCCAACATAG	118
2d6-8	CACCTAGTCCTCAATGCCAC	119
			2d6-9	GAGTCTTGCAGGGGTATCAC	120

< 9-3>is to each genotypic analysis respectively

For remaining 123 illustrative embodiments<9-1>Middle isolating genome DNA sample has been analyzed respectively and mainly has been found in the varient among the Aisa people ^*2A, ^*5, ^*2N, ^*10B, ^*14B, ^*18, ^*21B, ^*41A, ^*49, ^*52 draws ^*60 genotype.

A) analysis of CYP2D6*5

In order to confirm said CYP2D6*5 genotype, the primer in the use table 27 carries out PCR.Said 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 the CYP2D6*5 varient, 3.5kb PCR product has increased.

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

B) analysis of CYP2D6*2N

In order to confirm said CYP2D6*2N genotype, the primer in the use table 28 carries out PCR.Said 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 the CYP2D6*2N varient, produced 7.8kb PCR product.

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

C) analysis of CYP2D6*2 and * 41

Except that-1584C＞G varient, the CYP2D6*2 genotype comprises identical varient (1235A＞G with the CYP2D6*41 genotype;-740C＞T;-678G＞A; Gene converts 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 converts the said varient of CYP2D7 into AS-PCR method (Johanson, Molecular 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 the primer 10B that contains with reference to 125 with the primer 9 that contains with reference to 129.If said CYP2D6*2 genotype and CYP2D6*41 genotype are normal, then only with contain with reference to 129 primer 9 with contain with reference to 130 primer 10 be combined into performing PCR the time just can produce amplified production.Therefore, can change through confirm the said gene that converts the CYP2D7 gene in the introne 1 into the existence that is combined into the amplified production that performing PCR produces of said primer 9 and said primer 10B.

Said 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.Sequencing primer with in the table 29 is right-and 1584C＞G varient carries out the tetra-sodium order-checking.Confirmed-1584G be the CYP2D6*2 genotype and-1584C is the CYP2D6*41 genotype.

The gene order of [table 29] primer

D) CYP2D6*10B, * 14, * 18 and * 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 said CYP2D6*10B, * 14, * 18 and * 49 genotype.The primer is as shown in Table 30, shown in experiment condition such as the table 31.

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

[table 31] to 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?or?654	MwoI	348+258+39	272+258+85+39
					*49	486	Sau3A?I	347+142	286+142+61

E) CYP2D6*21, * 52 and * 60 genotypic analyses

Through PCR-tetra-sodium sequencing analysis said CYP2D6*21, * 52 and * 60 genotype.The gene order of the primer that said analysis is used is shown in table 32.

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

Based on the gene sequencing of disclosed CYP2D6 gene among the gene pool accession number M33388 data that produce among illustrative embodiments < 9-2>and < 9-3 >.Shown in each allelic frequency such as the 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 said 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 such as table 34 shown in.In table 34, " 1 " is meant wild-type and " 2 " refer to varient.

[table 34]

Illustrative embodiments 10: the selection of htSNP and checking

Confirm 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 confirm effectively said genotype, said htSNP has the details of haplotype.Essential accurately each haplotype of mark of said htSNP, and comprise various combinations.With SNPtagger software ( Http:// www.well.ox.ac.uk/～xiayi/haplotype/) select said htSNP to make up that is optimum set of tags.Shown in the instance such as Figure 34～39 of selected htSNP combination.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 confirm mutual two times of types that do not overlap and genotype.Then, confirm the htSNP combination.

According to said checking result, can confirm 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 confirms that genotypic said analysis is absolutely correct.

Illustrative embodiments 11: SNaPshot analyzes

Selected htSNP makes up and carries out the SNaPshot analysis in the usage example property 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 confirmed.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 confirmed 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
			htSNP1	SNP2，7，19	-1426C＞T
htSNP2	SNP3，6，10，27，30，31	3877G＞A
			htSNP3	SNP8，9，12，13，14，15，16，17，18，25	2850C＞T
htSNP4	SNP20
			1611T＞A
htSNP5	SNP22				1758G＞A
		htSNP6	SNP23			1887insTA
htSNP7	SNP24			2573insC
		htSNP8	SNP26
	2988G＞A
		htSNP9	SNP29	4125-4133ins9bp
htSNP10	SNP32				Deletion
		htSNP11	SNP33	Repeat

Use with illustrative embodiments < 9-2>in identical method increase said CYP2D6 gene to produce about 6.7kb product.For confirming CYP2D6*5, use the primer CYP2D6_3 (5 ' ACCTCTCTGGGCCCTCAGGGA-3 ') that contains reference 154 and contain primer 3 ' 2D6*5 amplification CYP-REP-Del with reference to 123.Said 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 possibly not influence said 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 1 multiple pre-reaction mixture of μ L SNaPshot (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 process totally 10 μ L reactants.Then, the PCR of said 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

Reaction mixes 10 μ L reactants after accomplishing 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 said sample 95 ℃ of reactions with 3130 genetic analysis appearance (ABI).Income analysis result as shown in Figure 40.

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; Producing 3.3kb PCR product, but use the Dup-F_2 that contains with reference to 155 (5 '-CCTCACCACAGGACTGGCCACC-3 ') and the Dup-R that contains reference 156 (5 '-CACGTGCAGGGCACCTAGAT-3 ') here with same procedure amplification CYP-REP-Dup.For from said 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, said 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; Process the reactant of 10 μ L, to carry out the SNaPshot reaction under the same conditions.Analyze said reactant with 3100 genetic analysis appearance.Analytical results is shown in figure 41.

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

Through 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.

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

Illustrative embodiments 12: confirm genotype with gene chip

The making of < 12-1>Zip coding chip

1) making of probe

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

[table 37]

2) point sample of probe is with fixing

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

The making of < 12-2>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 said 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

Said long PCR product, 1X AmpliTaq damping fluid, 0.2mMdNTP, each primer of 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 said 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.Said 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), 1X Band Doctor (Solgent) and the deionized water of the said 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 said 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 said 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 said damping fluid, and at 42 ℃ of incubations more than 30 minutes.Then said chip is cleaned 3 times with zero(ppm) 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 said 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 L 3X SSC then above that.Be loaded into said sample said on the chip of prehybridization, said 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 said chip is cleaned 1 time then, lasts 10 minutes, thereafter in room temperature clean 4 times each 1 minute.With the chip that cleaned put into canalis spinalis immediately and with separating centrifuge 800rpm drying 5 minutes.

6) analyze

The said chip that uses output wavelength that Axon makes to prepare for the GenePix 4100B scanner scanning of about 650nm.Fluorescence signal intensity with GenePix Pro 6.0 software analysis scanning images.Shown in said analytical results such as Figure 46 and the table 44.

[table 44]

The varient of the CYP2D6 gene of analyzing with said gene chip as a result, is with identical by the varient of sequencing analysis.

<PXR>

Illustrative embodiments 13: confirm high beauty PXR gene genotype

The amplification of < 13-1>PXR gene

Behind 54 health objects collection blood, use the genomic dna separating kit of Qiagen manufactured from blood, to isolate DNA.Analyzed the complete genome sequence of PXR gene with ABI 3130XL genetic analysis appearance.As a result, in 18 kinds of functional varients having found up to the present to report 6 kinds.Said PXR gene comprises 9 exons, and it is long to be approximately 38kb.With the exon that contains functional varient is the center, and said PXR gene is divided into 10 fragments, so that said fragment is carried out PCR.The PCR the primer is shown in table 45.The A, 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

Shown in the size such as table 46 of the position of said primer and PCR product.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

Shown in the reaction conditions such as table 47 that each PCR fragment is corresponding.

[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

The order-checking of < 13-2>PCR product

Analyzed the gene order of each PCR product of illustrative embodiments < 13-1>generation with automated DNA sequenator and the primer that contains reference 131～150.

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.Shown 22 SNP in the table 48, the functional varient of reporting is used the # mark.

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

SNP	The position	The rs numbering	Frequency (%)
				-25564G＞A	The upper reaches	rs12721602	1.9
#-25385C＞T	The upper reaches	rs3814055	16.7
				-24840A＞G	The upper reaches		0.9
-24622A＞T	The upper reaches		2.8
				-24446C＞A	The upper reaches	rs2276705	2.8
-24381A＞C	The upper reaches		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

Shown in table 48,7 kinds of varients of said 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 confirming the functional varient of PXR

In illustrative embodiments 13, studied 6 kinds of functional varients functional of PXR gene, said functional varient possibly 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 said PXR gene possibly influence the activity of PXR gene.Can be with the specifying information of the minimum mark inspection haplotype that generates.Said 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) 14 haplotypes selecting in the illustrative embodiments 14 are checked order selecting said 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 confirmed in the said combination of analyze finding with Matlab software (7.1 editions, Mai Siwoke softcom limited, the U.S.).Analytical results is used for confirming said combination.

According to said checking result, two times of types and genotype need not overlap and just can be able to confirm.In other words, the htSNP combination of selecting according to the present invention is different, and confirms that genotypic said 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 said PXR gene function property.Use the DNA of object to carry out PCR, amplified production is carried out SNaPshot analyze as template.The used primer of said PCR is shown in table 50.

[table 50]

The reaction conditions of said 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 said 4 through amplification PCR products.Not possibly 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 said product of handling through enzyme and the primer in the table 52 to carry out PCR and process multiple SNaPshot reactant.Shown in said multiple SNaPshot reactant and PCR reaction conditions such as table 53 and the 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 accomplished, 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 said mixture with 3130XL genetic analysis appearance (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 said 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 according to the 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 said 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 said UGT1A gene of amplification in the step 2 with known 3130X genetic analysis appearance (Applied Biosystems).(the gene pool accession number: gene order NT_005120) compares with analytical results and wild-type UGT1A gene.Shown in result such as table 56 and the 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 polymorphum 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 confirmed, 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 two mutants.

[table 58]

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

Selected the polymorphum of metabolic UGT1A1, UGT1A6 and the UGT1A9 gene of known participation resistive connection bowelcancer medicine Rinotecan based on the polymorphum 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 polymorphum 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, said object contain the UGT1A gene wild-type, contain the allelic varient of heterozygosis and contain the allelic varient that isozygotys.

Use 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 said 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.Here, the total amount of said 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 following condition.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 is mixed with 0.2 μ L LIZ120 (ABI) and 9.3 μ L Hi-Di methane amides and places 96 orifice plates.Response sample is analyzed by 3130X genetic analysis appearance (Applied Biosystems) 95 ℃ of reactions 2 minutes then.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 according to the 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 said-39insTA is confirmed by the order-checking of PCR-tetra-sodium.Be used for shown in the gene order such as table 61 of primer of said analysis.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 [Clin Chem., Jul; 49 (7): 1182-1185,2003].

The PCR product that more specifically, 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 PCR product that is generated and 37 μ L pH 7.6 ^TMHigh Performance AmershamBioscience) mixes.Then, said 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 2mM MgAc with the 1X annealing buffer of 100 μ L pH 7.6 ₂) mix and place 96 orifice plates.Handle said 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 polymorphum, 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 said 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 through order-checking in the size that has confirmed the peak and type and the exemplary embodiment 17 is identical.

Industrial applicibility

As stated; The analytical procedure of the functional varient of CYP1A2 of the present invention, CYP2A6, CYP2D6, PXR and UGT1A gene or the polymorphum relevant with drug susceptibility can be used for the polymorphum relevant with drug susceptibility of the functional varient or the UGT1A gene of definite CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene, and said method is used based on the optimum search combination of the polymorphum of still undetermined high beauty CYP1A2, CYP2A6, CYP2D6, PXR and UGT1A gene before.The present invention can be applied to confirm such as with 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 certain exemplary embodiment of the present invention and explains; But one skilled in the art will recognize that; Can under the situation that does not deviate from principle of the present invention and essence, change said illustrative embodiments, scope of the present invention limits in accompanying claims and equivalent.

Claims (17)

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

(a) collection of biological sample from object;

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

(c) carry out PCR with primer, this reaction uses the said 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) confirm haplotype by the gene order of the PCR product of having confirmed to have said varient in operation in (d); With

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

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

3. the said primer of the method for claim 1, wherein operating in (c) is selected from the primer that contains with reference to 76～89.

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

5. the method for claim 1, wherein the existence of said varient confirmed to comprise the gene order that contrasts said PCR product and the gene order of wild-type CYP2A6 gene in the operation (d).

6. the method for claim 1, said method also comprises repetitive operation (a)～(d).

7. confirm the method for human CYP2A6 gene genotype, said method comprises:

(a) collection of biological sample from object;

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

(c) carry out PCR with primer, this reaction uses the said 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), said varient comprises :-48T＞G; 13G＞A; 567C＞T; 2134A＞G; 3391T＞C; 6458A＞T; 6558T＞C; 6582G＞T; 6600G＞T; Be selected from a kind of among 6091C＞T, 5971G＞A and the 5983T＞G.

8. method as claimed in claim 7, wherein, the said biological specimen of collecting in the operation (a) is selected from blood, skin cells, myxocyte and hair.

9. method as claimed in claim 7, wherein, the said primer in the operation (c) is selected from the primer that contains with reference to 90,91,102 and 103.

10. method as claimed in claim 7, wherein, said operation (d) comprises the existence of definitive variation body, said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G.

11. method as claimed in claim 7, wherein, said operation (d) comprises the existence of definitive variation body, and said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G.

12. method as claimed in claim 7, wherein, said operation (d) comprises the existence of definitive variation body, and said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G.

13. method as claimed in claim 7, wherein, said operation (d) comprises the existence of definitive variation body, and said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G.

14. method as claimed in claim 7, wherein, said operation (d) comprises the existence of definitive variation body, and said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G.

15. method as claimed in claim 7, wherein, said operation (d) comprises the existence of definitive variation body, and said 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 a kind of among 6091C＞T, 5971G＞A and the 5983T＞G.

16. method as claimed in claim 7, wherein, confirming to comprise and analyze the existence of confirming said varient the existence of said varient in the operation (d) with SNaPshot.

17. method as claimed in claim 16, wherein, said SNaPshot analyzes with being selected from the primer that contains with reference in 92～101 the primer and carries out.

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