CN103436544A

CN103436544A - CYP2D6 gene segment containing 1745C>G mutant, protein segment coded by same and application thereof

Info

Publication number: CN103436544A
Application number: CN2013104010597A
Authority: CN
Inventors: 蔡剑平; 胡国新; 戴大鹏; 耿培武; 蔡杰
Original assignee: 蔡剑平
Priority date: 2013-09-05
Filing date: 2013-09-05
Publication date: 2013-12-11
Anticipated expiration: 2033-09-05
Also published as: CN103436544B

Abstract

The invention belongs to the field of biology and relates to a single base mutant at the 1745th site of CYP2D6 allele, wherein C at the site is mutated into G. The invention particularly relates to a nucleic acid segment containing the mutant site and a corresponding protein segment coded by the nucleic acid segment, a reagent and detection method for identifying the mutant site, application of the site, and especially application of identification of the site in medicine instruction.

Description

Comprise the 1745C>CYP2D6 gene fragment of G sudden change, coded protein fragments and application thereof

Technical field

The invention belongs to field of biology, relate to the single base mutation of the 1745th, CYP2D6 allelotrope.More specifically, the present invention relates to the nucleic acid fragment and the protein fragments of corresponding encoded, the reagent of identifying described mutational site, detection method and the application of this site of evaluation in direction of medication usage that comprise this mutational site.

Background technology

Cytochrome P 4502 D 6 (CYP2D6) is one of important member of CYP enzyme family.Its gene is positioned on No. 22 karyomit(e), comprises 9 exons, full length gene 9432bp(GenBank number of registration M33388, exon 1 is positioned at the 1620-5909 position).In human body, the amount of these cytopigment only accounts for 2%～4% of liver enzyme total amount, but participate in the metabolism of 20%～30% medicine clinically, medicine comprises thymoleptic, anti-arrhythmic, antipsychotic drug, anodyne, antitussive, antiemetic, antidiabetic drug and Bextra etc. like this, studies its metabolic polymorphism and has very important clinical value (referring to reference 1).

The CYP2D6 gene has the height polymorphism.So far, included in the NCBI snp database and surpassed 300 mutational sites.Allelotrope by the international name of CYP450 committee member name also has more than 150, also has in addition multiple newfound saltant type not yet by name (http://www.cypalleles.ki.se/cyp2d6.htm).Each allelotrope relates to point mutation, disappearance, insertion, rearrangement etc. in various degree, thereby affects the activity of CYP2D6, and then impact produces drug effect in various degree to the metabolic activity of medicine.Remove outside wild-type CYP2D6*1, more and the saltant type that clinical meaning is larger of at present research mainly comprises following 7 kinds: CYP2D6*2, * 3, * 4, * 5, * 10, * 17, * 41, wherein ethnic group distribute the widest, most study, Chinese population research data relatively the abundantest saltant type be CYP2D6*2(2850C T; 4180G > C) and CYP2D6*10(100C T; 4180G > C) (referring to reference 1,2,3).

According to current clinical studies show, this polymorphism of CYP2D6 gene is to cause the CYP2D6 enzymic activity in the significant major cause of interindividual variation, carry the very big difference that can cause curative effect of medication between the genotypic individuality of different CYP2D6, even produce serious poisonous side effect of medicine or treat insufficient.Therefore, research CYP2D6 gene pleiomorphism will provide important scientific basis (referring to reference 3,4) to clinical rational drug use to the impact of curative effect of medication.

Summary of the invention

The new single base mutation site that the purpose of this invention is to provide the CYP2D6 gene, the nucleic acid fragment that comprises this mutational site, the protein fragments of its coding and identify the application of this mutational site in medication guide.

First aspect of the present invention is to provide nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1745th corresponding to SEQ ID NO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, wherein the Nucleotide of the 1745th is G; Perhaps described nucleic acid fragment comprises the mutational site of the 492nd corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 492nd is G; The reverse complementary sequence that perhaps described nucleic acid fragment is above-mentioned nucleic acid fragment.

Second aspect of the present invention is to provide and contains corresponding to the 1745th of SEQ ID NO.1 or corresponding to the allelotrope fragment in the mutational site of the 492nd of SEQ ID NO.2 or the allele specific oligonucleotide of all or part of hybridization of its reverse complementary sequence, and wherein the Nucleotide in the mutational site of the 492nd of the 1745th of SEQ ID NO.1 the or SEQ ID NO.2 is G; Described allelotrope fragment is at least 10 continuous nucleotides or its reverse complementary sequence in the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

The 3rd aspect of the present invention is to provide for detection of and/or analyzes the test kit of single base mutation of the present invention, described test kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprise can be as the described single base mutation of primer amplification but do not comprise the nucleic acid fragment of this list base; Described single base is corresponding to the 1745th or the 492nd of SEQ ID NO.2 of SEQ ID NO.1.

The 4th aspect of the present invention is to provide nucleic acid fragment of the present invention or the application of oligonucleotide in detecting the CYP2D6 transgenation, and wherein said nucleic acid fragment or oligonucleotide are as probe or primer; Perhaps nucleic acid fragment of the present invention or oligonucleotide are for the preparation of the application of the medicine that detects the CYP2D6 transgenation; Perhaps nucleic acid fragment of the present invention or oligonucleotide are as the application of the check mark thing that detects the CYP2D6 transgenation.

The 5th aspect of the present invention is to provide a kind of medication guide, comprises the 1745th or the single base mutation of the 492nd of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2D6 gene in testing sample; According to the sudden change detected, adjust the dosage by the medicine of CYP2D6 metabolism.

The 6th aspect of the present invention is to provide the method for analysis of nucleic acids, described method comprise analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1745th or analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the Nucleotide of the 492nd.

The 7th aspect of the present invention is to provide CYP2D6 albumen or its fragment or varient, and described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the leucine of the 164th corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3.

The invention provides the CYP2D6 gene and the encoding sequence that comprise new single base mutation.This gene sports G(492C at the 492nd Nucleotide corresponding to SEQ ID NO.2 by C > G), thereby cause that the amino acid of its coding sports leucine by phenylalanine, corresponding to the leucine of the 164th of SEQ ID NO.3.The CYP2D6 albumen of this sudden change (called after F164L) is compared reduction to the metabolic activity of medicine with wild-type.This single base mutation has directive significance to the medication of the individuality that carries this mutational site.

The accompanying drawing explanation

Fig. 1 is the collection of illustrative plates that checks order of the heterozygote carrier corresponding to SEQ ID NO.1 sequence of the present invention in embodiment 1, and wherein the arrow indication is the 1745th Nucleotide of CYP2D6 gene;

Fig. 2 is the structural representation collection of illustrative plates of the double gene expression vector pIRES-Gluc-2D6 of embodiment 2 structures;

Fig. 3 is the CYP2D6.1(wild-type shown in embodiment 2), the CYP2D6.2(deficient mutants), the CYP2D6.10(deficient mutants) and F164L albumen of the present invention for the metabolic capacity detected result of Dextromethorphane Hbr, wherein * means p value<0.05;

Fig. 4 is the CYP2D6.1(wild-type shown in embodiment 2), the CYP2D6.2(deficient mutants), the CYP2D6.10(deficient mutants) and F164L albumen of the present invention detect collection of illustrative plates for the typical LC-MS/MS of Dextromethorphane Hbr metabolism; Arrow indication place is respectively the fignal center of substrate Dextromethorphane Hbr (right side) and meta-bolites demethyl Dextromethorphane Hbr (left side) thereof; Wherein X-coordinate means retention time, and the retention time of Dextromethorphane Hbr and demethyl Dextromethorphane Hbr is respectively 7.5 and 4.8 minutes, ordinate zou expression signal response value cps.

Embodiment

By following embodiment explanation the present invention, but content of the present invention is not limited to this.

As without other explanation, " nucleic acid fragment " of the present invention is comprised of Nucleotide or its analogue, can be the fragment of DNA, RNA or its analogue; Can be strand or two strands; It can be natural (as genomic) or synthetic.

In the present invention, " sudden change " refers at the gene detected, in the CYP2D6 gene, has the nucleotide site different from wild-type CYP2D6 gene order." mutational site " refers to the position that base is undergone mutation.In the present invention, described mutational site is corresponding to the 492nd in sequence shown in the 1745th of sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

International P450 allelotrope NK is about stipulating in CYP2D6 allelotrope naming rule: using first base A of initiator codon ATG in CYP2D6 genomic dna reference sequences (GenBank number of registration M33388) as allelic the 1st of CYP2D6 (first base A of initiator codon is positioned at the 1620th of M33388 sequence), the definite catastrophe point of the present invention is positioned at allelic the 1745th of CYP2D6 (SEQ ID NO.1).

Content of the present invention relates to the nonsynonymous mutation of CYP2D6 gene.Because this mutational site is arranged in the encoding sequence of gene, therefore, those skilled in the art are known, and described mutational site both can show in genomic dna, also can in encoding sequence (being CDS), show.Those skilled in the art, according to detected sample, can be detected this mutational site on genomic dna or mRNA level.In the application, SEQ ID NO.1 is that wherein the 1745th is the mutational site the present invention relates to according to the CYP2D6 allelotrope sequence of the present invention of the stipulative definition of international P450 allelotrope NK.SEQ ID NO.2 is the cDNA sequence with the CYP2D6 gene in described mutational site, and wherein the 492nd is the mutational site the present invention relates to.Those skilled in the art are known, in this article, and corresponding to the 1745th site of SEQ ID NO.1 with use mutually corresponding to the 492nd the site synonym of SEQ ID NO.2.

In the present invention, " allele-specific " refer to specifically and allelotrope hybridization, as hybridized, make the 492nd Nucleotide of identifying corresponding to sequence shown in the 1745th of sequence shown in SEQ ID NO.1 or SEQ ID NO.2 under rigorous condition, is G.

In the present invention, Nucleotide and amino acid whose abbreviation adopt abbreviation mode well known in the art, and as A in Nucleotide means VITAMIN B4, G means guanine, and C means cytosine(Cyt), and T means thymus pyrimidine.In amino acid, A means L-Ala, and R means arginine, and N means l-asparagine, D means aspartic acid, and C means halfcystine, and Q means glutamine, and E means L-glutamic acid, G means glycine, and H means Histidine, and I means Isoleucine, and L means leucine, K means Methionin, and M means methionine(Met), and F means phenylalanine, P means proline(Pro), and S means Serine, and T means Threonine, W means tryptophane, and Y means tyrosine, and V means α-amino-isovaleric acid.

Content of the present invention is based on the new single base mutation site of CYP2D6 gene.Described mutational site is the coding region that is positioned at the CYP2D6 gene, and corresponding to the 492nd of SEQ ID NO.2, this site sports G by the C of wild-type; In addition, the 164th by the albumen of the CYP2D6 genes encoding of this sudden change sports leucine (F164L) by phenylalanine.

Aspect first, the invention provides nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1745th corresponding to SEQ ID NO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, and wherein the Nucleotide of the 1745th is G; Perhaps described nucleic acid fragment comprises the mutational site of the 492nd corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 492nd is G; It is perhaps the reverse complementary sequence of above-mentioned nucleic acid fragment.

In one embodiment, the length of described nucleic acid fragment can be as 10-100,101-200, a 201-500 or 501-1000 Nucleotide.Preferably, the length of described nucleic acid fragment is 10-20,21-30,31-40,41-50,51-60, a 61-100 or 101-300 Nucleotide.

Described mutational site can be positioned at any position of described nucleic acid fragment.

In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.1.

In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.2.

In other embodiments, described nucleic acid fragment can be the sequence shown in SEQ ID NO.14-18.

In one embodiment, described oligonucleotide is as probe.Described probe can be under rigorous condition and the target sequence specific hybrid that comprises mutational site.It is known to those skilled in the art that described probe does not need and the target sequence complete complementary, if can with the target sequence specific hybridization.In preferred embodiments, described hybridization conditions can meet make probe only with the target sequence specific hybrid.The length of described probe can be 5-100 Nucleotide, as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,40,50,60,70,80,90 or 100 Nucleotide.Described mutational site can appear at any position of probe.In a preferred embodiment, described mutational site appears at center or about center of probe sequence.

In another embodiment, described oligonucleotide is with coaching the synthetic primer of DNA, sequencing primer or synthetic primer etc. as known in the art.Described primer does not need and the template complete complementary, but should be with the complementary hybridization of template to instruct DNA synthetic.The length of described primer can be 15-40 length of nucleotides, is preferably 18,19,20,21,22,23,24,25,26,27,28,29 or 30 Nucleotide.Described mutational site can appear at any position of described primer; Preferably, described mutational site appears at 3 ' end of described primer.

Some preferred embodiment in, described oligonucleotide is the sequence as shown in SEQ ID NO.19-23.

Based on this, the 3rd aspect of the present invention be to provide for detection of and/or the test kit of analysis list base mutation, described test kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprise can be as the described single base mutation of primer amplification but do not comprise the nucleic acid fragment of this list base; Described single base is corresponding to the 1745th or the 492nd of SEQ ID NO.2 of SEQ ID NO.1.Preferably, described test kit comprises the sequence fragment shown in SEQ ID NO.4 and/or SEQ ID NO.5 and/or SEQ ID NO.10.

The 4th aspect of the present invention is to provide the application for detection of the CYP2D6 transgenation of nucleic acid fragment of the present invention or oligonucleotide, and wherein said nucleic acid fragment or oligonucleotide are as probe or primer; Perhaps nucleic acid fragment of the present invention or oligonucleotide are for the preparation of the application of the medicine that detects the CYP2D6 transgenation; Perhaps nucleic acid fragment of the present invention or oligonucleotide are as the application of the check mark thing that detects the CYP2D6 transgenation.

The 5th aspect of the present invention is to provide medication guide, comprises the 1745th or the base of the 492nd of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2D6 gene in testing sample.When the CYP2D6 gene detected is G in the site of the 492nd corresponding to the 1745th of SEQ ID NO.1 or SEQ ID NO.2, adjust accordingly the dosage through the medicine of CYP2D6 metabolism.In specific embodiment, when the CYP2D6 gene is G in the site of the 492nd of SEQ ID NO.2, the CYP2D6 protease activity of this genes encoding descends, therefore need to adjust the dosage through the medicine of CYP2D6 metabolism, assigns as follows dose.

The medicine through the CYP2D6 metabolism described in the present invention comprises: beta-blockers, Proprasylyte, metoprolol, H-56/28, bufuralol, timolol, bunitrolol, carvedilol, alprenolol, nebivolol; Anti-arrhythmic, encainide, sparteine, Tamboar, Propafenone, aprindine, mexiletine, encainide, procainamide; Antihypertensive drug, Debrisoquine, Indoramine; Antianginal, perhexiline, terodiline; Anodyne, U-26225A; Anti-spiritual medicine, chlorpromazine, trilafon, haloperidol, risperidone, thioridazine, zuclopenthixol, Aripiprazole; Tricyclic antidepressant, amitriptyline, imipramine, chlorimipramine, Desipramine, nortriptyline; Other thymoleptic, fluoxetine, paroxetine, Venlafaxine, fluvoxamine, amiflamine, mianserin, brofaromine, maprotiline, tomoxetine, amphetamine, citalopram, fluvoxamine, Minaprine, duloxetine, moclobemide; Medicine for the treatment of cough and asthma, dihydrocodeine, Ethylmorphine, morphine monomethyl ether, Dextromethorphane Hbr; Antidiabetic drug, phenformin; Other, Toldrin, metoclopramide, Tomoxetine hydrochloride, Isomeride, ondansetron, amphetamines, lignocaine, ondansetron, Phenacetin, triphen chloramines, dexfenfluramine, promethazine.

In one embodiment, described method can be restriction fragment length polymorphism analysis (RFLP).Those skilled in the art can be according to the present invention the content design experiment Nucleotide of the 492nd of take in the nucleic acid of sequence of the Nucleotide of the 1745th in the nucleic acid of the sequence of analyzing SEQ ID NO.1 or SEQ ID NO.2 whether be G.

In another embodiment, described method can be sequencing, comprise and separate and measure the nucleotide sequence from genomic dna or RNA, analyze wherein comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1745th or whether comprise corresponding to the Nucleotide corresponding to the 492nd in the nucleic acid of the sequence of SEQ ID NO.2 be G.Sequencing can be any available sequence measurement known in the art.Sequencing primer can be designed according to those skilled in the art's general knowledge, as the design of the upstream and downstream appropriate position in site to be detected primer, contains the fragment in this site to be measured with expanding packet, thereby judges the Nucleotide in this site.Also can adopt oligonucleotide of the present invention as primer sequence.

In another embodiment, described method is to utilize the method for probe hybridization, in the identification and detection sample, comprise specifically corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1745th or whether comprise corresponding to the Nucleotide corresponding to the 492nd in the nucleic acid of the sequence of SEQ ID NO.2 be G; The probe adopted in described method is oligonucleotide of the present invention.For example, isolate nucleic acid from testing sample, under allowing the condition of probe and the specific target sequence hybridization that may exist in nucleic acid, probe is contacted with nucleic acid; The hybridization that can be detected can realize by using the probe of being crossed by detectable reagent mark; For example, form the enzyme that can detect product with radio isotope, fluorescence dye or energy catalysis and carry out label probe.Label probe, detect with label probe whether to have the method for target sequence in sample be all well-known to those skilled in the art.

In a kind of concrete embodiment, provide with Taqman probe SNP detection method and detect the method corresponding to the Nucleotide of the 1745th of SEQ ID NO.1, comprising:

1) the design primer comprises the PCR product of the 1745th corresponding to SEQ ID NO.1 for specific amplification, designs two Taqman-MGB probes, respectively for C and the G allelotrope of the 1745th corresponding to SEQ ID NO.1 simultaneously.

The design of primers principle is:

(1) choose should be at the conservative section of gene for sequence;

(2) avoid primer self or and primer between form pairing continuously more than 4 or 4, avoid primer self to form the pili annulati card structure;

(3) primer length is at 18 to 24 Nucleotide;

(4) the Tm value is at 55-65 ℃, and GC content is at 40%-60%;

(5) the Tm value between primer differs and avoids over 2 ℃;

(6) 3 ' of primer end avoids using base A, and 3 ' end of primer avoids occurring base consecutive identical more than 3 or 3;

(7) pcr amplified fragment length is at 50bp-150bp;

(8) last 5 Nucleotide of primer end can not have G and the C that surpasses 2.

Taqman MGB probe design principle is:

(1) 5 ' of probe end avoids occurring G;

(2) the Tm value should be 65-67 ℃;

(3) shorten Taqman MGB probe, but probe length is no less than 13bp as far as possible;

(4) avoid the base, especially the G base that duplicate as far as possible, avoid occurring that the G more than 4 or 4 repeats;

(5) mutational site of probe is placed on as far as possible to the place of centre 1/3.

Fluorophor can adopt FAM, VIC etc. to carry out two allelotrope of mark.

2) utilize above-mentioned primer and probe, sample to be tested is carried out to real-time quantitative PCR.

The PCR condition: 95 ℃ of denaturations enter 30 amplification cycles after 10 minutes: 92 ℃ of sex change 12 seconds, 60 ℃ of annealing and extend 1 minute (this stage is detected fluorescent signal).

3) data analysis.

Analyze experimental result, according to the power of two kinds of fluorescence of sample, judge whether sample to be tested CYP2D6 gene exists 1745C the G sudden change.

In the present invention, described sample can be any sample that comprises nucleic acid, as blood; Preferred described sample comes from the people.Described nucleic acid can be DNA or coding RNA, is preferably genomic dna.It is target compound that the method for analysis of nucleic acids of the present invention can be take DNA or RNA.Those skilled in the art are known, when take DNA when detecting target compound, analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1745th, the probe used or primer are according to the sequences Design of SEQ ID NO.1; When take RNA when detecting target compound, analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the Nucleotide of the 492nd, the probe used or primer are according to the sequences Design of SEQ ID NO.2.

The 7th aspect of the present invention is to provide CYP2D6 albumen or its fragment or varient, and described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the leucine of the 164th corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3, as 10-20,21-50 or 51-100 amino acid.

Below will further illustrate the present invention by specific embodiment, but following specific embodiment is only for exemplary purpose.

Embodiment 1: the evaluation in the mutational site that people CYP2D6 gene is new

In the present embodiment, gather Normal Occlusion of Han People healthy population blood sample, extract the genomic dna in blood, the design sequencing primer carries out sequence amplification, order-checking to 9 exons of CYP2D6 gene, analyzes its CYP2D6 gene and whether has mutational site.

1) extract DNA:

Take 5ml vein EDTA anticoagulated blood sample from the measured; Then according to common salting-out process and/or adopt special DNA extraction test kit (purchased from the DNA extraction test kit of U.S. Omega company) to extract the genomic dna of blood sample to be measured.

2) pcr amplification:

Design of amplification primers, increased to 9 exon sequences of CYP2D6 gene in the genome DNA sample obtained.Described amplimer to sequence in Table 1.

Adopt 30 μ L PCR reaction systems, comprising: 1 * GC PCR damping fluid, 1.5mM MgCl ₂, the genomic dna of 100ng, upstream and downstream primer be the LATaqDNA polysaccharase 1.5U that 0.2 μ M, dNTP are 0.2mM, TaKaRa company.Use the GeneAmp PCR System9700 amplification instrument amplification of American AB I company.The pcr amplification loop parameter is as follows: 94 ℃ of denaturations 2 minutes, and 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 68 ℃ are extended 3 minutes, extend 3 minutes after 35 circulations again.Primer sequence information is in Table 1.

Table 1: amplimer is to sequence information

3) purifying amplified production:

By the amplified production that obtains according to MultiScreen HTS ^tMthe operation instruction of test kit (U.S. Millipore company), the DNA that carries out the purpose band reclaims purifying.

4) order-checking:

The product of take after reclaiming is template, use sequencing primer according to the PCR reaction of being checked order of BigDye Terminator v3.1 sequencing kit (American AB I company) operation instruction, purifying amplified production after reaction finishes, used the Prism3730XL type gene sequencer of American AB I company to be separated the sequence with the interpretation amplified production.The sequencing primer sequence information is in Table 2.

Table 2: sequencing primer sequence information table

Zone	Sequencing primer (5 '-3 ')
		Exons 1	AGGAAGCAGGGGCAAGAAC(SEQ?ID?NO.8)
Exon 2	CGCCCTCTCTGCCCAGC(SEQ?ID?NO.9)
		Wai Xianzi3 &4	TTGGAGTGGGTGGTGGA(SEQ?ID?NO.10)
Exon 5&6	AGGARGTYAGGCTTACAGGA(SEQ?ID?NO.11)
		Exon 7	GCACAGGCTTGACCAGGAT(SEQ?ID?NO.12)
Exon 8&9	TGTTTGGTGGCAGGGGTCC(SEQ?ID?NO.13)

5) data analysis:

The sequence and the wild-type CYP2D6*1 sequence (GenBank number of registration M33388) that record are compared.

By compare of analysis, find to have in 2129 routine experimenters 1 people's CYP2D6 genomic dna to carry a kind of brand-new mutation type, the Nucleotide that corresponding CYP2D6 is allelic the 1745th becomes G(as shown in Figure 1 by C, wherein S means C and G heterozygosis).This sudden change is positioned at the 3rd exon of CYP2D6 gene, and catastrophe point is positioned at the 492nd of cDNA, infers accordingly in the protein of this CYP2D6 genes encoding, and the 164th amino acids sports leucine (L) by phenylalanine (F).

The present embodiment has exemplarily provided the method for identifying the new mutant site.Those skilled in the art can clearly learn according to foregoing the method comprised corresponding to the 1745th Nucleotide of SEQ ID NO.1 that detects specifically in testing sample: the nucleic acid in sample separation, carry out amplified reaction under corresponding experiment condition in the present embodiment, primer is used primer pair SEQ ID NO.4 and 5; With sequencing primer SEQ ID NO.10, the product of amplification is checked order; Sequencing result and wild-type result are compared, analyzed the Nucleotide corresponding to the 1745th site of SEQ ID NO.1.

Embodiment 2: the vitro enzyme metabolic activity is analyzed

According to existing result of study, wild-type (* 1 type) is all higher to the metabolic activity of various medicines, and the metabolic activity of * 2 types has obvious decline than the metabolic activity of wild-type, and the metabolic activity of * 10 types is than * 2 types lower (referring to document 6,7).Therefore, existing a kind of like this common recognition in the art: the expressed enzyme of same genotype can represent the metabolic activity to other substrate medicine to the metabolic activity of specific substrate.Thereby, according to a certain genotype expressed enzyme to specific substrate metabolic activity data can analogize the expressed enzyme of this genotype to the metabolic activity of other substrate medicine (as, the metabolic activity of the enzyme that the metabolic activity of enzyme that can this genotype is expressed and wild-type are expressed compares).

In the present embodiment, according to above-mentioned mutational site, take wild-type CYP2D6(*1) gene is template, rite-directed mutagenesis the Nucleotide (by C, becoming G) of the 492nd of CYP2D6 gene coding region, the expression vector of construction expression saltant type CYP2D6 albumen (called after F164L), after transfection 293FT cell, add CYP2D6 specific probe substrate---Dextromethorphane Hbr, after hatching, detect the In vitro metabolism activity of this saltant type CYP2D6 albumen by analyzing substrate and meta-bolites amount, with judgement, with wild-type CYP2D6.1, compare, whether its enzymatic metabolic activity changes.This experimental design be similar to we at CYP2C9 new mutant body medicine in-vitro the operation steps during for activation analysis, the most authoritative magazine Pharmacogenomics J approval (document 5 sees reference) by the International Pharmaceutical genomics field of its science.

1) vivoexpression of CYP2D6 varient

Take and comprise wild-type CYP2D6(*1) plasmid vector of full-length cDNA (buying from Thermo Scientific company) is template, utilizes side-directed mutagenesis to obtain respectively the cDNA of CYP2D6*2, CYP2D6*10 and F164L mutant of the present invention.Side-directed mutagenesis is techniques well known, and those skilled in the art, according to definite template and target, can know beyond all doubtly and how complete this step.By sequence is correct after testing each goal gene and reference gene Gluc(secretor type luciferase, its translation product can be secreted in substratum, and fluorescent signal detected by the particular agent box; Its skeleton carrier is pIRES pGluc-Basic, purchased from NEB company, article No. N8082S) be connected to respectively double gene expression vector pIRES(purchased from Clontech company, article No. 631605) in A, B multiple clone site, a kind of goal gene and reference gene are positioned under same CMV promotor control, finally obtain double gene expression vector pIRES-Gluc-2D6(structural representation collection of illustrative plates and see Fig. 2).Build and to comprise respectively CYP2D6(*1) four kinds of CYP2D6 double gene expression vectors of the cDNA of cDNA, CYP2D6*2cDNA, CYP2D6*10cDNA and F164L mutant of the present invention.

By 5 * 10 ⁵individual 293FT cell (people's renal epithelial cell source, purchased from Invitrogen company) evenly is laid on 6 orifice plates; After incubated overnight, utilize liposome lip2000(Invitrogen company) transfection 2 μ g plasmid vector pIRES-Gluc-2D6, to express various target protein CYP2D6 and internal reference Protein G luc.Carry out the western hybridization check after transfection 24h, determine above-mentioned four kinds of target proteins correction.

Cell cultures and liposome transfection are techniques well known, and the illustration method provided with reference to Invitrogen company just can carry out.

2) viable cell incubated in vitro probe medicament

Continue to cultivate after 24 hours, by single hole cell dissociation in 6 orifice plates resuspended to 300 μ l substratum (use EP pipe carries out).Use the classical probe medicine Dextromethorphane Hbr of CYP2D6 (buying from U.S. Sigma company) to detect the activity of this enzyme, the final concentration of this medicine is 20 μ M.Under 37 ℃, at 5%CO ₂in incubator, 3h is hatched in the 300rpm concussion.From CO ₂take out culture in incubator, adding the 0.1MNaOH of 20 μ L and shaking vortex 1min(reason is that Dextromethorphane Hbr is weak base, and solution, with existing with molecular conformation after the NaOH alkalization, is extracted out thereby easily enter organic phase in next step extraction process).Add 800 μ L glacial acetic acid ethyl esters, concussion vortex 2min is placed on-40 ℃ of refrigerator 30min, until lower floor is freezing.Take out sample, under 4 ℃, with the centrifugal 10min of 12000g.The upper strata ethyl acetate is transferred in new EP pipe, on 37 ℃ of Nitrogen evaporators, ethyl acetate is dried up.Add 200 μ L separately initial flow phase solution redissolve, after concussion vortex 1min with the centrifugal 5min of 12000g.Supernatant is transferred in sample injection bottle after using initial flow phase solution by the 1:10 dilution.

3) LC-MS/MS detects

After abstraction purification, sample carries out the LC-MS/MS detection on 1260-6410 type instrument (U.S. Agilent company), and HPLC adopts ZORBAX SB-C18 post (150mm * 4.6mm, diameter 5 μ m).

The Dextromethorphane Hbr testing conditions:

Chromatographic condition

Column temperature: 30 ℃

Chromatographic column: ZORBAX SB-C18 (Agilent, 5 μ m, 4.6 * 150mm)

Sampling volume: 2 μ L

Flow velocity: 0.6mL/min

Working time: 8min

Table 3: Dextromethorphane Hbr mobile phase ratio

Time (min)	10mM ammonium acetate (%, v/v)	Methyl alcohol (%, v/v)
			0.01	40	60
0.5	5	95
			4	5	95
4.01	40	60
			8	40	60

The mass spectrum condition:

Electron spray(ES) (ESI) ion source (positive ion), many reaction detection scanning (MRM) patterns;

Ionogenic temperature (TEM): 300 ℃;

Curtain gas speed: 11L/min;

Capillary voltage: 4000V;

Dextromethorphane Hbr Q1/Q3:272.2/213.1; 272.2/147.1;

Impact energy=30;

The cracked voltage of parent ion=140;

Demethyl Dextromethorphane Hbr Q1/Q3:258.2-157.1; 258.2-133.1;

Impact energy=35;

The cracked voltage of parent ion=135.

4) LC-MS/MS detects data analysis

2500,1250,500,250,100,20ng/mL prepare the Dextromethorphane Hbr of different gradient concentrations and corresponding meta-bolites demethyl Dextromethorphane Hbr (Dextromethorphane Hbr:; The demethyl Dextromethorphane Hbr: 1500,750,300,150,50,25ng/mL), generate typical curve after utilizing LC-MS to detect, and in order to detect the metabolism situation of various CYP2D6 albumen to Dextromethorphane Hbr, then use [product/(product+substrate)] to mean the metabolic rate of this probe medicine, after internal reference Gluc data calibration, mean the enzymic activity of mutein with the ratio of the metabolic rate of the metabolic rate of saltant type and wild-type.Every kind of albumen repeats respectively 3 times for the experiment of Dextromethorphane Hbr, and the rear statistics of averaging is respectively in Table 4 and Fig. 3; Various albumen the results are shown in Figure 4 to the typical mass spectrometric detection of Dextromethorphane Hbr.The metabolic capacity of CYP2D6 varient is stronger, the peak area at substrate (Dextromethorphane Hbr) peak will be less, and the peak area at product (demethyl Dextromethorphane Hbr) peak will be larger, as can be seen from the figure, the metabolic capacity of new varient F164L obviously reduces than wild-type, be less than typical mutant CYP2D6.2, but be greater than again typical mutant CYP2D6.10 simultaneously.

Table 4:293FT cell is hatched the enzymic activity result of probe medicament Dextromethorphane Hbr

* refer to the relative value with wild-type CYP2D6.1

* p value<0.05

Detected result shows, with respect to wild-type CYP2D6.1 type, known typical deficient mutants CYP2D6.2 is about 13% to the metabolic activity decline of Dextromethorphane Hbr, the metabolic activity of another typical defect type mutant CYP2D6.10 obviously descend (reduction amplitude approximately 88%), this result and existing document are basically identical, show that the data of our vitro detection system acquisition have very high confidence level (referring to reference 7,8).

Utilize this vitro detection system to find out: the F164L mutant is wild-type to the metabolic activity of probe medicament Dextromethorphane Hbr 76.29%.Statistical analysis shows, with wild-type, compares, and the metabolic activity of F164L mutant descends, and has significant difference, and the metabolic activity of pointing out this sudden change can cause expressed enzyme obviously reduces.Therefore, in practice, need to consider suitably to regulate on dosage carrying this genotypic individuality, as the usage quantity that reduces medicine and the generation of avoiding adverse drug reaction.This medicine (as imipramine, amitriptyline etc.) larger for individual difference by the medicine adjustment of gene targeting is even more important.

Reference

1.Zhou?SF.Polymorphism?of?Human?Cytochrome?P4502D6and?Its?Clinical?Significance:Part?I.Clinical?Pharmacokinetics.2009,48(11):689-723.

2.Qin?S,Shen?L,Zhang?A,et?al.Systematic?polymorphism?analysis?of?the?CYP2D6gene?in?four?different?geographical?Han?populations?in?mainland?China.Genomics.2008,92(3):152-158.

3.ZHOU?SF.Polymorphism?of?Human?Cytochrome?P4502D6and?Its?Clinical?Significance?Part?II.Clinical?pharmacokinetics.2009,48(12):761-804.

4. Xu Yan is tender, Gong Sen, and Ji Hongyan, wait .CYP2D6 gene pleiomorphism and clinical meaning thereof. medical Leader .2012,31 (10): 1337-1340.

5.Dai?DP,Xu?RA,Hu?LM,Wang?SH,Geng?PW,Yang?JF,et?al.CYP2C9polymorphism?analysis?in?Han?Chinese?populations:building?the?largest?allele?frequency?database.Pharmacogenomics?J.2013,DOI:10.1038/tpj.2013.2.

6.Wennerholm?A,Johansson?I,Hidestrand?M,Bertilsson?L,Gustafsson?LL,Ingelman-Sundberg?M.Characterization?of?the?CYP2D6*29allele?commonly?present?in?a?black?Tanzanian?population?causing?reduced?catalytic?activity.Pharmacogenetics2001,11:417-427.

7.Sakuyama?K,Sasaki?T,Ujiie?S,Obata?K,Mizugaki?M,Ishikawa?M?et?al.Functional?characterization?of17CYP2D6allelic?variants(CYP2D6.2,10,14A-B,18,27,36,39,47-51,53-55,and57).Drug?Metab?Dispos.2008,36:2460-2467.

Sequence:

SEQ ID NO.1: allelotrope sequence

ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGCAGAGGTGCTGAGGCTCCCCTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAGAAGCCAGGCTGAGAAGGGGAAGCAGGTTTGGGGGACGTCCTGGAGAAGGGCATTTATACATGGCATGAAGGACTGGATTTTCCAAAGGCCAAGGAAGAGTAGGGCAAGGGCCTGGAGGTGGAGCTGGACTTGGCAGTGGGCATGCAAGCCCATTGGGCAACATATGTTATGGAGTACAAAGTCCCTTCTGCTGACACCAGAAGGAAAGGCCTTGGGAATGGAAGATGAGTTAGTCCTGAGTGCCGTTTAAATCACGAAATCGAGGATGAAGGGGGTGCAGTGACCCGGTTCAAACCTTTTGCACTGTGGGTCCTCGGGCCTCACTGCCTCACCGGCATGGACCATCATCTGGGAATGGGATGCTAACTGGGGCCTCTCGGCAATTTTGGTGACTCTTGCAAGGTCATACCTGGGTGACGCATCCAAACTGAGTTCCTCCATCACAGAAGGTGTGACCCCCACCCCCGCCCCACGATCAGGAGGCTGGGTCTCCTCCTTCCACCTGCTCACTCCTGGTAGCCCCGGGGGTCGTCCAAGGTTCAAATAGGACTAGGACCTGTAGTCTGGGGTGATCCTGGCTTGACAAGAGGCCCTGACCCTCCCTCTGCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCCTGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGCAAGCAGCGGTGGGGACAGAGACAGATTTCCGTGGGACCCGGGTGGGTGATGACCGTAGTCCGAGCTGGGCAGAGAGGGCGCGGGGTCGTGGACATGAAACAGGCCAGCGAGTGGGGACAGCGGGCCAAGAAACCACCTGCACTAGGGAGGTGTGAGCATGGGGACGAGGGCGGGGCTTGTGACGAGTGGGCGGGGCCACTGCCGAGACCTGGCAGGAGCCCAATGGGTGAGCGTGGCGCATTTCCCAGCTGGAATCCGGTGTCGAAGTGGGGGCGGGGACCGCACCTGTGCTGTAAGCTCAGTGTGGGTGGCGCGGGGCCCGCGGGGTCTTCCCTGAGTGCAAAGGCGGTCAGGGTGGGCAGAGACGAGGTGGGGCAAAGCCTGCCCCAGCCAAGGGAGCAAGGTGGATGCACAAAGAGTGGGCCCTGTGACCAGCTGGACAGAGCCAGGGACTGCGGGAGACCAGGGGGAGCATAGGGTTGGAGTGGGTGGTGGATGGTGGGGCTAATGCCTTCATGGCCACGCGCACGTGCCCGTCCCACCCCCAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGCGCGAGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGGCCTGGGCAAGAAGTCGCTGGAGCAGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTGGCCAACCACTCCGGTGGGTGATGGGCAGAAGGGCACAAAGCGGGAACTGGGAAGGCGGGGGACGGGGAAGGCGACCCCTTACCCGCATCTCCCACCCCCAGGACGCCCCTTTCGCCCCAACGGTCTCTTGGACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCGGAGCGAGAGACCGAGGAGTCTCTGCAGGGCGAGCTCCCGAGAGGTGCCGGGGCTGGACTGGGGCCTCGGAAGAGCAGGATTTGCATAGATGGGTTTGGGAAAGGACATTCCAGGAGACCCCACTGTAAGAAGGGCCTGGAGGAGGAGGGGACATCTCAGACATGGTCGTGGGAGAGGTGTGCCCGGGTCAGGGGGCACCAGGAGAGGCCAAGGACTCTGTACCTCCTATCCACGTCAGAGATTTCGATTTTAGGTTTCTCCTCTGGGCAAGGAGAGAGGGTGGAGGCTGGCACTTGGGGAGGGACTTGGTGAGGTCAGTGGTAAGGACAGGCAGGCCCTGGGTCTACCTGGAGATGGCTGGGGCCTGAGACTTGTCCAGGTGAACGCAGAGCACAGGAGGGATTGAGACCCCGTTCTGTCTGGTGTAGGTGCTGAATGCTGTCCCCGTCCTCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGACCCAGCTGGATGAGCTGCTAACTGAGCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGTGAGAGTGGCTGCCACGGTGGGGGGCAAGGGTGGTGGGTTGAGCGTCCCAGGAGGAATGAGGGGAGGCTGGGCAAAAGGTTGGACCAGTGCATCACCCGGCGAGCCGCATCTGGGCTGACAGGTGCAGAATTGGAGGTCATTTGGGGGCTACCCCGTTCTGTCCCGAGTATGCTCTCGGCCCTGCTCAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTGCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGTGAGCCCATCTGGGAAACAGTGCAGGGGCCGAGGGAGGAAGGGTACAGGCGGGGGCCCATGAACTTTGCTGGGACACCCGGGGCTCCAAGCACAGGCTTGACCAGGATCCTGTAAGCCTGACCTCCTCCAACATAGGAGGCAAGAAGGAGTGTCAGGGCCGGACCCCCTGGGTGCTGACCCATTGTGGGGACGCATGTCTGTCCAGGCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGATTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGTGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGTAGGCCTGGCGCCCTCCTCACCCCAGCTCAGCACCAGCACCTGGTGATAGCCCCAGCATGGCTACTGCCAGGTGGGCCCACTCTAGGAACCCTGGCCACCTAGTCCTCAATGCCACCACACTGACTGTCCCCACTTGGGTGGGGGGTCCAGAGTATAGGCAGGGCTGGCCTGTCCATCCAGAGCCCCCGTCTAGTGGGGAGACAAACCAGGACCTGCCAGAATGTTGGAGGACCCAACGCCTGCAGGGAGAGGGGGCAGTGTGGGTGCCTCTGAGAGGTGTGACTGCGCCCTGCTGTGGGGTCGGAGAGGGTACTGTGGAGCTTCTCGGGCGCAGGACTAGTTGACAGAGTCCAGCTGTGTGCCAGGCAGTGTGTGTCCCCCGTGTGTTTGGTGGCAGGGGTCCCAGCATCCTAGAGTCCAGTCCCCACTCTCACCCTGCATCTCCTGCCCAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCTCAGCAGGTGCCTGTGGGGAGCCCGGCTCCCTGTCCCCTTCCGTGGAGTCTTGCAGGGGTATCACCCAGGAGCCAGGCTCACTGACGCCCCTCCCCTCCCCACAGGCCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAGCACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAG

SEQ ID NO.2: encoding sequence

ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCCTGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGCGCGAGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGGCCTGGGCAAGAAGTCGCTGGAGCAGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTGGCCAACCACTCCGGACGCCCCTTTCGCCCCAACGGTCTCTTGGACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCTGAATGCTGTCCCCGTCCTCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGACCCAGCTGGATGAGCTGCTAACTGAGCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTGCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGATTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGTGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCTCAGCAGGCCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAGCACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAG

SEQ ID NO.3: protein sequence

MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYPPGPLPLPGLGNLLHVDFQNTPYCFDQLRRRFGDVFSLQLAWTPVVVLNGLAAVREALVTHGEDTADRPPVPITQILGFGPRSQGVFLARYGPAWREQRRFSVSTLRNLGLGKKSLEQWVTEEAACLCAALANHSGRPFRPNGLLDKAVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLLHIPALAGKVLRFQKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDENLRIVVADLFSAGMVTTSTTLAWGLLLMILHPDVQRRVQQEIDDVIGQVRRPEMGDQAHMPYTTAVIHEVQRFGDIVPLGVTHMTSRDIEVQGFRIPKGTTLITNLSSVLKDEAVWEKPFRFHPEHFLDAQGHFVKPEAFLPFSAGRRACLGEPLARMELFLFFTSLLQHFSFSVPTGQPRPSHHGVFAFLVSPSPYELCAVPR

SEQ ID NO.14: nucleic acid fragment

TGCCGCCTTGGCCAACCACT

SEQ ID NO.15: nucleic acid fragment

CTTTGTGCCGCCTTGGCCAACCACTCCGGA

SEQ ID NO.16: nucleic acid fragment

CCTGCCTTTGTGCCGCCTTGGCCAACCACTCCGGACGCCC

SEQ ID NO.17: nucleic acid fragment

GGCCGCCTGCCTTTGTGCCGCCTTGGCCAACCACTCCGGACGCCCCTTTC

SEQ ID NO.18: nucleic acid fragment

GCCTGCCTTTGTGCCGCCTTGGCC

SEQ ID NO.19: oligonucleotide fragment

CCGGAGTGGTTGGCC

SEQ ID NO.20: oligonucleotide fragment

CGTCCGGAGTGGTTGGCCAAG

SEQ ID NO.21: oligonucleotide fragment

GAAAGGGGCGTCCGGAGTGGTTGGCC

SEQ ID NO.22: oligonucleotide fragment

GGTTGGCCAAGGCGG

SEQ ID NO.23: oligonucleotide fragment

GAGTGGTTGGCCAAGGCGGCACA

Claims

1. nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1745th corresponding to SEQ ID NO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, and wherein the Nucleotide of the 1745th is G; Perhaps described nucleic acid fragment comprises the mutational site of the 492nd corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 492nd is G; It is perhaps the reverse complementary sequence of above-mentioned nucleic acid fragment.

2. nucleic acid fragment according to claim 1, is characterized in that, the length of described nucleic acid fragment is 10-100,101-200, a 201-500 or 501-1000 Nucleotide; Preferably, the length of described nucleic acid fragment is 10-20,21-30,31-40,41-50,51-60, a 61-100 or 101-300 Nucleotide; Further preferably, described nucleic acid fragment be SEQ ID NO.1,2 or 14-18 shown in sequence.

3. allele specific oligonucleotide, described oligonucleotide with contain corresponding to the 1745th of SEQ ID NO.1 or corresponding to allelotrope fragment or all or part of hybridization of its reverse complementary sequence in the mutational site of the 492nd of SEQ ID NO.2, wherein the Nucleotide in the mutational site of the 492nd of the 1745th of SEQ ID NO.1 the or SEQ ID NO.2 is G; Described allelotrope fragment is at least 10 continuous nucleotides or its reverse complementary sequence in the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

4. allele specific oligonucleotide according to claim 3, is characterized in that, described oligonucleotide is probe or primer; Preferably, when described oligonucleotide is probe, the length of described oligonucleotide is 5-100 Nucleotide; When described oligonucleotide is primer, the length of described oligonucleotide is 15-40 Nucleotide; Preferably, when described oligonucleotide is probe, described mutational site is positioned at center or about center of probe sequence; When described oligonucleotide is primer, described mutational site is positioned at 3 ' end of primer.

5. allele specific oligonucleotide according to claim 4, is characterized in that, described oligonucleotide is the sequence as shown in SEQ ID NO.19-23.

One kind for detection of and/or the test kit of analysis list base mutation, comprise nucleic acid fragment according to claim 1 and 2 or according to the described allele specific oligonucleotide of claim 3-5 any one, or comprise can be as the described single base mutation of primer amplification but do not comprise the nucleic acid fragment of this list base; Described single base is corresponding to the 1745th or the 492nd of SEQ ID NO.2 of SEQ ID NO.1; Preferably, described test kit comprises the sequence fragment shown in SEQ ID NO.4 and/or SEQ ID NO.5 and/or SEQ ID NO.10.

7. nucleic acid fragment according to claim 1 and 2 or the application in detecting the CYP2D6 transgenation according to the described allele specific oligonucleotide of claim 3-5 any one, wherein said nucleic acid fragment or oligonucleotide are as probe or primer; The perhaps application in the medicine of preparation detection CYP2D6 transgenation; Perhaps be used as the application of the check mark thing that detects the CYP2D6 transgenation.

8. a medication guide, comprise the 1745th or the base of the 492nd of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2D6 gene in testing sample, when the base in described site is G, adjusts the dosage through the medicine of CYP2D6 metabolism.

9. the method for an analysis of nucleic acids, described method comprise analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1745th or analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the Nucleotide of the 492nd; Preferably, described method is sequencing, restriction fragment length polymorphism analysis or probe hybridization method.

10.CYP2D6 albumen or its fragment or varient, described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the leucine of the 164th corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3.