CN103451210B

CN103451210B - Including 2943G > A sudden change CYP2D6 genetic fragment, coded protein fragments and application thereof

Info

Publication number: CN103451210B
Application number: CN201310403498.1A
Authority: CN
Inventors: 蔡剑平; 胡国新; 戴大鹏; 耿培武; 蔡杰
Original assignee: Individual
Current assignee: Beijing Hospital
Priority date: 2013-09-06
Filing date: 2013-09-06
Publication date: 2016-11-09
Anticipated expiration: 2033-09-06
Also published as: CN103451210A

Abstract

The invention belongs to field of biology, relate to the single base mutation of the 2943rd, CYP2D6 allele, described site is sported A by G.In particular it relates to the nucleic acid fragment comprising this mutational site and the protein fragments of corresponding encoded thereof, identify the application in the reagent in described mutational site, detection method and this site, particularly identify the application in direction of medication usage of this site.

Description

Including 2943G > A sudden change CYP2D6 genetic fragment, coded protein fragments and Its application

Technical field

The invention belongs to field of biology, relate to the single base mutation of the 2943rd, CYP2D6 allele.More specifically, The present invention relates to comprise the nucleic acid fragment in this mutational site and the protein fragments of corresponding encoded thereof, identify described mutational site The application in direction of medication usage of this site of reagent, detection method and qualification.

Background technology

Cytochrome P 4502 D 6 (CYP2D6) is one of important member of CYP enzyme family.Its gene is positioned at No. 22 dyeing On body, including 9 exons, full length gene 9432bp(GenBank number of registration M33388, exon 1 is positioned at 1620-5909 Position).In human body, the amount of this cytochrome only accounts for the 2%～4% of liver enzyme total amount, but participates in the generation of 20%～30% medicine clinically Thanking, such medicine includes antidepressants, anti-arrhythmic, psychosis, analgesic, antitussive, Bendectin, anti-diabetic Medicine and Bextra etc., study its metabolic polymorphism and have highly important clinical value (seeing list of references 1).

CYP2D6 gene has height polymorphism.So far, NCBI snp database has been included more than 300 Mutational site.Also more than 150 are had by the allele of CYP450 Intemational Nomenclature committee member name, the most multiple newfound Saltant type is not yet named (http://www.cypalleles.ki.se/cyp2d6.htm).The different journey of each allele Degree relate to point mutation, lack, insert, rearrangement etc., thus affect the activity of CYP2D6, and then the metabolic activity that impact is to medicine, Produce drug influence in various degree.Remove outside wild type CYP2D6*1, the sudden change that research is more and clinical meaning is bigger at present Type mainly includes following 7 kinds: CYP2D6*2, * 3, * 4, * 5, * 10, * 17, * 41, wherein ethnic group distribution is the widest, most study, in The abundantest saltant type of state's crowd's research data is CYP2D6*2(2850C > T；4180G > C) and CYP2D6*10(100C > T；4180G > C) (seeing list of references 1,2,3).

According to current clinical studies show, this polymorphism of CYP2D6 gene is to cause CYP2D6 enzymatic activity at individuality Between the main cause of significant difference, carry the huge difference that can cause curative effect of medication between the individuality of different CYP2D6 genotype, Even produce serious poisonous side effect of medicine or treat insufficient.Therefore, research CYP2D6 gene pleiomorphism is to curative effect of medication Impact will provide important scientific basis (seeing list of references 3,4) to clinical rational drug use.

Summary of the invention

It is an object of the invention to provide the new single base mutation site of CYP2D6 gene, comprise the nucleic acid in this mutational site Fragment, its coding protein fragments and identify the application in medication guide of this mutational site.

The first aspect of the invention is to provide nucleic acid fragment, and described nucleic acid fragment comprises corresponding to SEQ ID NO.1's The mutational site of the 2943rd, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, wherein The nucleotide of the 2943rd is A；Or described nucleic acid fragment comprises the mutational site of the 979th corresponding to SEQ ID NO.2, And be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, wherein the nucleotide of the 979th is A； Or the reverse complementary sequence that described nucleic acid fragment is above-mentioned nucleic acid fragment.

The second aspect of the invention is to provide and contains the 2943rd corresponding to SEQ ID NO.1 or corresponding to SEQ The equipotential that the allele fragment in the mutational site of the 979th of ID NO.2 or its reverse complementary sequence completely or partially hybridize Gene specific oligonucleotides, the wherein mutational site of the 979th of the 2943rd or SEQ ID NO.2 of SEQ ID NO.1 Nucleotide be A；Described allele fragment be in the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 extremely Few 10 continuous nucleotides or its reverse complementary sequence.

The third aspect of the invention is to provide the test kit of the single base mutation for detecting and/or analyze the present invention, Described test kit comprises nucleic acid fragment or the allele specific oligonucleotide of the present invention, or comprise can be as primer amplification Described single base mutation but do not comprise the nucleic acid fragment of this list base；Described single base is corresponding to the 2943rd of SEQ ID NO.1 the Position or the 979th of SEQ ID NO.2.

The fourth aspect of the invention is to provide the nucleic acid fragment of the present invention or oligonucleotide and dashes forward at detection CYP2D6 gene Application in change, wherein said nucleic acid fragment or oligonucleotide are used as probe or primer；Or the nucleic acid fragment of the present invention or widow Nucleotide is for preparing the application of the medicine of detection CYP2D6 gene mutation；Or the nucleic acid fragment of the present invention or oligonucleotide are used Make the application of the check mark thing of detection CYP2D6 gene mutation.

The fifth aspect of the invention is to provide a kind of medication guide, including CYP2D6 gene right in detection testing sample Should be in the single base mutation of the 979th of the 2943rd of SEQ ID NO.1 or SEQ ID NO.2；According to the sudden change detected, Adjust by the dosage of the medicine of CYP2D6 metabolism.

The sixth aspect of the invention is to provide the method analyzing nucleic acid, and described method includes analyzing the bag in testing sample Containing corresponding to the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the nucleotide of the 2943rd or analyzing comprising in testing sample Corresponding to the nucleic acid of the sequence of SEQ ID NO.2 corresponds to the nucleotide of the 979th.

The seventh aspect of the invention is to provide CYP2D6 albumen or its fragment or variant, and described protein sequence is SEQ Sequence shown in ID NO.3；Described fragment or variant comprise the methionine of the 327th corresponding to SEQ ID NO.3, and At least 10 continuous amino acids for the aminoacid sequence shown in SEQ ID NO.3.

The invention provides the CYP2D6 gene and coded sequence comprising new single base mutation.This gene is corresponding to 979th nucleotide of SEQ ID NO.2 is sported A(979G by G > A), thus cause its aminoacid encoded to be dashed forward by valine Become methionine, i.e. corresponding to the methionine of the 327th of SEQ ID NO.3.The CYP2D6 albumen of this sudden change is (named V327M) the metabolic activity reduction compared with wild type to medicine.This single base mutation individual use to carrying this mutational site Medical instrument has directive significance.

Accompanying drawing explanation

Fig. 1 is heterozygous carriers's Sequencing chromatogram of the SEQ ID NO.1 sequence corresponding to the present invention in embodiment 1, Wherein arrow indication is the 2943rd nucleotide of CYP2D6 gene；

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

Fig. 3 is the CYP2D6.1(wild type shown in embodiment 2), CYP2D6.2(deficient mutants), CYP2D6.10 The V327M albumen of (deficient mutants) and the present invention for the metabolic capacity testing result of dextromethorphan and bufuralol, its Middle * represents p value < 0.05；

Fig. 4 is the CYP2D6.1(wild type shown in embodiment 2), CYP2D6.2(deficient mutants), CYP2D6.10 The V327M albumen of (deficient mutants) and the present invention detects collection of illustrative plates for the typical LC-MS/MS of dextromethorphan metabolism；Arrow Substrates dextromethorphan (right) and the signal peak of metabolite demethyl dextromethorphan (left) thereof it is respectively at indication；Wherein abscissa Representing retention time, the retention time of dextromethorphan and demethyl dextromethorphan is respectively 7.5 and 4.8 minutes, and vertical coordinate represents Signal response value cps；

Fig. 5 is the CYP2D6.1(wild type shown in embodiment 2), CYP2D6.2(deficient mutants), CYP2D6.10 The V327M albumen of (deficient mutants) and the present invention detects collection of illustrative plates for the typical LC-MS/MS of bufuralol metabolism；Arrow Substrate bufuralol (right) and the signal peak of metabolite 1 '-hydroxyl bufuralol (left) thereof it is respectively at indication；The most horizontal seat Mark represents retention time, and the retention time of bufuralol and 1 '-hydroxyl bufuralol is respectively 6.9 and 5.1 minutes, vertical coordinate table Show signal response value cps.

Detailed description of the invention

By following detailed description of the invention, the present invention is described, but present invention is not limited to this.

As without other illustrate, " nucleic acid fragment " of the present invention is made up of nucleotide or its analog, can be DNA, RNA or The fragment of its analog；Can be strand or double-strand；Can be natural (such as genome) or synthesis.

In the present invention, " sudden change " refers to the gene in detection, i.e. exist and wild type CYP2D6 gene sequence in CYP2D6 gene Arrange different nucleotide sites." mutational site " refers to the position that base is undergone mutation.In the present invention, described mutational site is right Should be in the 979th in sequence shown in the 2943rd of sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

World P450 allele NK specifies about in CYP2D6 allele naming rule: with CYP2D6 base Because of in group DNA reference sequences (GenBank number of registration M33388) first base A of start codon ATG as CYP2D6 etc. 1st the 1620th of M33388 sequence (first base A of start codon be positioned at) of position gene, then present invention determine that Catastrophe point is positioned at allelic 2943rd of CYP2D6 (SEQ ID NO.1).

Present invention relates to the nonsynonymous mutation of CYP2D6 gene.Owing to this mutational site is positioned at the coded sequence of gene In, therefore, skilled person will appreciate that, described mutational site both can show in genomic DNA, it is also possible at code sequence Performance in row (i.e. CDS).Those skilled in the art, can be to this on genomic DNA or mRNA level in-site according to detected sample Mutational site is detected.In the application, SEQ ID NO.1 is that the regulation according to world P450 allele NK is fixed The CYP2D6 allelic sequences of the present invention of justice, wherein the 2943rd is the mutational site that the present invention relates to.SEQ ID NO.2 Being the cDNA sequence of the CYP2D6 gene with described mutational site, wherein the 979th is the mutational site that the present invention relates to.This Skilled person understands, in this article, corresponding to the 2943rd site of SEQ ID NO.1 with corresponding to SEQ ID NO.2 The 979th site synonym use mutually.

In the present invention, " allele-specific " refers to hybridize, as carried out under high stringency conditions with allele specifically Hybridization so that identify the 979th corresponding to sequence shown in the 2943rd or SEQ ID NO.2 of sequence shown in SEQ ID NO.1 Position nucleotide is A.

In the present invention, nucleotide and amino acid whose abbreviation use abbreviation mode well known in the art, as in nucleotide, A represents Adenine, G represents that guanine, C represent that cytosine, T represent thymus pyrimidine.In aminoacid, A represents that alanine, R represent essence ammonia Acid, N represents that agedoite, D represent that aspartic acid, C represent that cysteine, Q represent that glutamine, E represent that glutamic acid, G represent Glycine, H represents that histidine, I represent that isoleucine, L represent that leucine, K represent that lysine, M represent that methionine, F represent benzene Alanine, P represents that proline, S represent that serine, T represent that threonine, W represent that tryptophan, Y represent that tyrosine, V represent figured silk fabrics ammonia Acid.

Present disclosure is new single base mutation site based on CYP2D6 gene.Described mutational site is in In the coding region of CYP2D6 gene, corresponding to the 979th of SEQ ID NO.2, this site is sported A by the G of wild type；This Outward, it is methionine (V327M) by the 327th of the albumen of the CYP2D6 gene code of this sudden change by valine mutation.

At first aspect, the present invention provides nucleic acid fragment, described nucleic acid fragment to comprise corresponding to SEQ ID NO.1 The mutational site of 2943, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, Qi Zhong The nucleotide of 2943 is A；Or described nucleic acid fragment comprises the mutational site of the 979th corresponding to SEQ ID NO.2, and Being at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, wherein the nucleotide of the 979th is A；Or Person is the reverse complementary sequence of above-mentioned nucleic acid fragment.

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

Described mutational site may be located 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 used as probe.Described probe can under high stringency conditions with bag Target sequence specific hybrid containing mutational site.It is known to those skilled in the art that described probe need not the most mutual with target sequence Mend, as long as can be with target sequence specific hybridization.In preferred embodiments, described hybridization conditions can meet and makes probe only With target sequence specific hybrid.The length of described probe can be 5-100 nucleotide, such 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 occur in any position of probe.Preferred embodiment In, described mutational site occurs in center or the about center of probe sequence.

In another embodiment, the described oligonucleotide primer of the DNA synthesis that coaches, survey as known in the art Sequence primer or synthetic primer etc..Described primer need not and template complete complementary, but it should with template Complementary hybridization to instruct DNA Synthesis.The length of described primer can be 15-40 length of nucleotides, it is therefore preferable to 18,19,20,21,22,23,24,25, 26,27,28,29 or 30 nucleotide.Described mutational site can occur in any position of described primer；Preferably, described Mutational site occurs in 3 ' ends of described primer.

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

Based on this, the third aspect of the invention is to provide the test kit for detecting and/or analyze single base mutation, institute State nucleic acid fragment or allele specific oligonucleotide that test kit comprises the present invention, or comprise can be as primer amplification institute State single base mutation but do not comprise the nucleic acid fragment of this list base；Described single base is corresponding to the 2943rd of SEQ ID NO.1 Or the 979th of SEQ ID NO.2.Preferably, described test kit comprise SEQ ID NO.6 and/or SEQ ID NO.7 and/or Sequence fragment shown in SEQ ID NO.11.

The fourth aspect of the invention is to provide the nucleic acid fragment of the present invention or oligonucleotide for detecting CYP2D6 gene The application of sudden change, wherein said nucleic acid fragment or oligonucleotide are used as probe or primer；Or the nucleic acid fragment of the present invention or widow Nucleotide is for preparing the application of the medicine of detection CYP2D6 gene mutation；Or the nucleic acid fragment of the present invention or oligonucleotide are used Make the application of the check mark thing of detection CYP2D6 gene mutation.

The fifth aspect of the invention is to provide medication guide, including the corresponding to of CYP2D6 gene in detection testing sample The 2943rd of SEQ ID NO.1 or the base of the 979th of SEQ ID NO.2.When the CYP2D6 gene detected is in correspondence When the site of the 979th of the 2943rd or SEQ ID NO.2 of SEQ ID NO.1 is A, adjust accordingly through CYP2D6 The dosage of the medicine of metabolism.In the particular embodiment, when CYP2D6 gene is in the site of the 979th of SEQ ID NO.2 During for A, the CYP2D6 proteinase activity of this gene code declines, therefore needs to adjust the dosage of the medicine through CYP2D6 metabolism, Assign dose as follows.

The heretofore described medicine through CYP2D6 metabolism includes: beta-blocker, Propranolol, metoprolol, alkene Third Luo Er, bufuralol, timolol, bunitrolol, carvedilol, alprenolol, nebivolol；Anti-arrhythmic, English Card amine, sparteine, flecainide, Propafenone, aprindine, mexiletine, encainide, procainamide；Antihypertensive, isoquinoline Guanidine, indoramine；Antianginal, perhexiline, terodiline；Analgesic, tramadol；Anti-spirit medicine, chlorpromazine, perphenazine, Haloperidol, risperidone, thioridazine, zuclopenthixol, Aripiprazole；Tricyclic antidepressant, amitriptyline, imipramine, chlorine Imipramine, desipramine, nortriptyline；Other antidepressants, fluoxetine, paroxetine, venlafaxine, fluvoxamine, Ah rice Husband's amine, mianserin, brofaromine, maprotiline, tomoxetine, amfetamine, citalopram, fluvoxamine, Minaprine, degree Duloxetine, moclobemide；Medicine for the treatment of cough and asthma, dihydrocodeine, ethylmorphine, codeine, dextromethorphan；Antidiabetic drug, benzene Second biguanide；Other, chlorphenamine, metoclopramide, atomoxetine, Isomeride, ondansetron, amphetamines, benefit card Cause, ondansetron, phenacetin, triphen chloramines, dexfenfluramine, promethazine.

In one embodiment, described method can be restriction fragment length polymorphism analysis (RFLP).This area Technical staff can be according to present invention contrived experiment with the 2943rd 's in the nucleic acid of the sequence of analysis SEQ ID NO.1 Whether the nucleotide of the 979th in the nucleic acid of the sequence of nucleotide or SEQ ID NO.2 is A.

In another embodiment, described method can be sequencing, including separating and measure from genomic DNA or The nucleotide sequence of RNA, analyze wherein comprise in the nucleic acid of the sequence corresponding to SEQ ID NO.1 corresponding to the core of the 2943rd Thuja acid or in comprising the nucleic acid of the sequence corresponding to SEQ ID NO.2 whether be A corresponding to the nucleotide of the 979th.Order-checking Method can be any available sequence measurement known in the art.Sequencing primer can enter according to the general knowledge of those skilled in the art Row design, as primer is designed in the upstream and downstream appropriate position in site to be detected, contains the fragment in this site to be measured with expanding packet, from And judge the nucleotide in this site.The oligonucleotide of the present invention can also be used as primer sequence.

In another embodiment, described method is the method utilizing probe to hybridize, specifically identification and detection sample In comprise in the nucleic acid of the sequence corresponding to SEQ ID NO.1 corresponding to the nucleotide of the 2943rd or comprise corresponding to SEQ In the nucleic acid of the sequence of ID NO.2, whether the nucleotide corresponding to the 979th is A；The probe used in described method is this Bright oligonucleotide.Such as, from testing sample, isolate nucleic acid, allow probe and specific target that may be present in nucleic acid Probe is made to contact with nucleic acid under conditions of sequence hybridization；The hybridization that can be detected can be by using by detectable reagent labelling The probe crossed realizes；Such as, maybe can be catalyzed with radiosiotope, fluorescent dye to be formed and can detect the enzyme of product and carry out labelling and visit Pin.Label probe, it is all well known to those skilled in the art with whether label probe detection sample exists the method for target sequence 's.

In a kind of specific embodiment, it is provided that detect corresponding to SEQ ID NO.1 with Taqman probe SNP detection method The method of nucleotide of the 2943rd, including:

1) design primer comprises the PCR primer of corresponding to SEQ ID NO.1 the 2943rd for specific amplification, simultaneously Design two Taqman-MGB probes, be respectively directed to G and the A allele of corresponding to SEQ ID NO.1 the 2943rd.

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 4 or more than 4 continuously pairings, it is to avoid primer self formation Pili annulati card structure；

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

(4) Tm value is at 55-65 DEG C, and G/C content is at 40%-60%；

(5) the Tm value difference between primer avoids exceeding 2 DEG C；

(6) 3 ' ends of primer are avoided using base A, and 3 ' ends of primer avoid the occurrence of 3 or more than 3 consecutive identical alkali Base；

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

(8) last 5 nucleotide of prime end can not have more than G and C of 2.

Taqman MGB probe design principle is:

(1) 5 ' ends of probe avoid the occurrence of G；

(2) Tm value should be 65-67 DEG C；

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

(4) avoid the occurrence of the base of repetition, especially G base, it is to avoid occur that the G of 4 or more than 4 repeats as far as possible Existing；

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

Fluorophor can use FAM, VIC etc. to carry out two allele of labelling.

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

PCR condition: 95 DEG C of denaturations enter 30 amplification cycles after 10 minutes: 92 DEG C of degeneration 12 seconds, 60 DEG C of annealing and prolonging Stretch 1 minute (this stage detection fluorescence signal).

3) data analysis.

Analyzing experimental result, strong and weak according to two kinds of fluorescence of sample judges whether sample to be tested CYP2D6 gene exists 2943G > A suddenlys change.

In the present invention, described sample can be any sample comprising nucleic acid, such as blood；The most described sample comes from People.Described nucleic acid can be DNA or coding RNA, preferably genomic DNA.The method analyzing nucleic acid of the present invention can be with DNA Or RNA is object.Skilled person will appreciate that, when with DNA for detection object, comprising in analysis testing sample is right Should be corresponding to the nucleotide of the 2943rd in the nucleic acid of the sequence of SEQ ID NO.1, the probe used or primer are according to SEQ The sequential design of ID NO.1；When with RNA for detection object, analyze comprising corresponding to SEQ ID NO.2 in testing sample Sequence nucleic acid in corresponding to the nucleotide of the 979th, the probe used or primer set according to the sequence of SEQ ID NO.2 Meter.

The seventh aspect of the invention is to provide CYP2D6 albumen or its fragment or variant, and described protein sequence is SEQ Sequence shown in ID NO.3；Described fragment or variant comprise the methionine of the 327th corresponding to SEQ ID NO.3, and are At least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3, such as 10-20,21-50 or 51-100 amino Acid.

The present invention will be further illustrated by specific embodiment below, but embodiment in detail below will be merely for exemplary Purpose.

Embodiment 1: the qualification in the mutational site that people's CYP2D6 gene is new

In the present embodiment, gathering Normal Occlusion of Han People healthy population blood sample, extract the genomic DNA in blood, design is surveyed Sequence primer carries out sequence amplification, order-checking to 9 exons of CYP2D6 gene, analyzes whether its CYP2D6 gene exists sudden change position Point.

1) DNA is extracted:

5ml vein EDTA anticoagulated blood sample is taked from measured；Then according to common salting out method and/or employing are special DNA extraction kit (purchased from the DNA extraction kit of Omega company of the U.S.) extract the genomic DNA of blood sample to be measured.

2) PCR amplification:

9 exon sequences of CYP2D6 gene in the genome DNA sample obtained are expanded by design amplimer Increase.Described amplimer is shown in Table 1 to sequence.

Use 30 μ L PCR reaction systems, including: 1 × GC PCR buffer, 1.5mM MgCl₂, the genome of 100ng DNA, upstream and downstream primer be 0.2 μM, dNTP be the LATaq archaeal dna polymerase 1.5U of 0.2mM, TaKaRa company.Use the U.S. The GeneAmp PCR System9700 amplification instrument amplification of ABI company.PCR amplification cycles parameter is as follows: 94 DEG C of denaturations 2 points Clock, 94 DEG C of degeneration 30 seconds, anneal 30 seconds for 60 DEG C, 68 DEG C extend 3 minutes, re-extend 3 minutes after 35 circulations.Primer sequence information It is shown in Table 1.

Table 1: amplimer is to sequence information

3) purification amplified production:

By the amplified production that obtains according to the use of MultiScreen HTSTM test kit (Millipore company of the U.S.) Illustrating, the DNA carrying out purpose band reclaims purification.

4) order-checking:

With the product after recovery as template, use sequencing primer according to BigDye Terminator v3.1 sequencing kit (American AB I company) operation instruction carries out PCR reaction of checking order, and reaction terminates rear purification amplified production, uses American AB I company Prism3730XL type gene sequencer carries out separating with the sequence of interpretation amplified production.Sequencing primer sequence information is shown in Table 2.

Table 2: sequencing primer sequence information table

Region	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 recorded is compared with wild type CYP2D6*1 sequence (GenBank number of registration M33388).

Analyzed by comparison, find 2129 example experimenters have the CYP2D6 genomic dna sequence of 1 people carry a kind of brand-new Mutation type, the nucleotide of allelic 2943rd corresponding of CYP2D6 is become A(from G as it is shown in figure 1, wherein R represents G and A heterozygosis).This sudden change is positioned at the 6th exon of CYP2D6 gene, and catastrophe point is positioned at cDNA the 979th, and inferring accordingly should In the protein of CYP2D6 gene code, the 327th amino acids is sported methionine (M) by valine (V).

The present embodiment exemplarily gives the method identifying new mutation site.Those skilled in the art are according to foregoing Can clearly learn and specifically detect the side comprising the 2943rd nucleotide corresponding to SEQ ID NO.1 in testing sample Method: separating the nucleic acid in sample, carry out amplified reaction under experiment condition corresponding in the present embodiment, primer uses primer pair SEQ ID NO.6 and 7；With sequencing primer SEQ ID NO.11, the product of amplification is checked order；By sequencing result and wild type Result comparison, analyzes the nucleotide in the 2943rd site corresponding to SEQ ID NO.1.

Embodiment 2: vitro enzyme metabolic activity is analyzed

According to existing result of study, wild type (* 1 type) is the highest to the metabolic activity of various medicines, and * 2 types Metabolic activity is decreased obviously than the metabolic activity of wild type, and the metabolic activity of * 10 types is lower than * 2 types (seeing document 6,7). Therefore, the most such a is known together: the metabolic activity to specific substrate of the enzyme expressed by same genotype The metabolic activity to other substrate medicine can be represented.Thus, according to the enzyme expressed by a certain genotype to specific substrate metabolism Activity data can analogize the enzyme expressed by this genotype (e.g., can be by this genotype institute to the metabolic activity of other substrate medicine The metabolic activity of the enzyme expressed compares with the metabolic activity of the enzyme expressed by wild type).

In the present embodiment, according to above-mentioned mutational site, with wild type CYP2D6(*1) gene as template, rite-directed mutagenesis The nucleotide (being become A from G) of the 979th of CYP2D6 gene coding region, construction expression saltant type CYP2D6 albumen is (named V327M) expression vector, after transfection 293FT cell, adds CYP2D6 Specific probe dextromethorphan and fourth furan Lip river You, after hatching, detect the In vitro metabolism activity of this saltant type CYP2D6 albumen by analysis substrate and metabolite amount, with Judging compared with wild type CYP2D6.1, whether its enzymatic metabolism activity changes.This experimental design be similar to we The external medicine of CYP2C9 new mutant is for operating procedure during activity analysis, and its science is by International Pharmaceutical genomics field The most authoritative magazine Pharmacogenomics J approves (see reference document 5).

1) vivoexpression of CYP2D6 variant

To comprise wild type CYP2D6(*1) plasmid vector of full-length cDNA (buy from Thermo Scientific company) For template, side-directed mutagenesis is utilized to obtain the cDNA of V327M mutant of CYP2D6*2, CYP2D6*10 and the present invention respectively. Side-directed mutagenesis is techniques well known, and those skilled in the art, can be beyond all doubt according to the template determined and target Known how this step.By glimmering to each genes of interest correct for sequence after testing and reference gene Gluc(secreting type Light element enzyme, its translation product can be secreted in culture medium, and fluorescence signal detected by particular agent box；Its skeleton carrier is PIRES pGluc-Basic, purchased from NEB company, article No. N8082S) it is respectively connecting to double gene expression vector pIRES(and is purchased from Clontech company, article No. 631605) A, B multiple clone site in, make a kind of genes of interest and reference gene be positioned at same CMV Under promoter controls, Fig. 2 is shown in by the final double gene expression vector pIRES-Gluc-2D6(structural representation collection of illustrative plates that obtains).Build respectively Including CYP2D6(*1) cDNA of the V327M mutant of cDNA, CYP2D6*2cDNA, CYP2D6*10cDNA and the present invention four Plant CYP2D6 double gene expression vector.

By 5 × 10⁵Individual 293FT cell (people's renal epithelial cell is originated, purchased from Invitrogen company) is uniformly laid on 6 holes Plate；After incubated overnight, utilize liposome lip2000(Invitrogen company) transfect 2 μ g plasmid vector pIRES-Gluc-2D6, To express various destination protein CYP2D6 and internal reference Protein G luc.Carry out western hybridization check after transfection 24h, determine above-mentioned Four kinds of destination proteins are the most correctly expressed.

Cell is cultivated and liposome transfection is techniques well known, the illustration method provided with reference to Invitrogen company Just can carry out.

2) living cells incubated in vitro probe medicament

Continue to cultivate after 24 hours, by single hole cell dissociation in 6 orifice plates and resuspended (use EP pipe to 300 μ l culture medium Carry out).Two kinds of CYP2D6 classics probe medicine dextromethorphans (buying from Sigma Co., USA) and bufuralol is used (to buy certainly Toronto Research Chemicals company of Canada) detect the activity of this enzyme, the final concentration of two kinds of medicines is respectively 20 μ M and 10 μMs.At 37 DEG C, at 5%CO₂In incubator, 3h is hatched in 300rpm concussion.From CO₂Incubator takes out culture, adds The 0.1MNaOH of 20 μ L also shakes vortex 1min(reason and is bufuralol and dextromethorphan is all weak base, and solution NaOH alkalizes After will exist with molecular conformation, in next step extraction process, be easily accessible organic facies thus be extracted out).Add 800 μ L Glacial acetic acid ethyl ester, concussion vortex 2min is placed on-40 DEG C of refrigerator 30min, until lower floor's frost.Take out sample, at 4 DEG C, with 12000g is centrifuged 10min.Upper strata ethyl acetate is transferred in new EP pipe, ethyl acetate is dried up by 37 DEG C of Nitrogen evaporators. Add 200 μ L each initial flow phase solution to redissolve, after concussion vortex 1min, be centrifuged 5min with 12000g.Supernatant uses initial Flowing phase solution is transferred in sample injection bottle after diluting by 1:10.

3) LC-MS/MS detection

After abstraction purification, sample carries out LC-MS/MS detection on 1260-6410 type instrument (Agilent company of the U.S.), HPLC uses ZORBAX SB-C18 post (150mm × 4.6mm, diameter 5 μm).

Dextromethorphan testing conditions:

Chromatographic condition

Column temperature: 30 DEG C

Chromatographic column: ZORBAX SB-C18 (Agilent, 5 μm, 4.6 × 150mm)

Sampling volume: 2 μ L

Flow velocity: 0.6mL/min

Operation time: 8min

Table 3: dextromethorphan mobile phase ratio

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

Mass Spectrometry Conditions:

Electron spray (ESI) ion source (cation), many reaction detection scanning (MRM) pattern；

Ionogenic temperature (TEM): 300 DEG C；

Curtain gas speed: 11L/min；

Capillary voltage: 4000V；

Dextromethorphan Q1/Q3:272.2/213.1；272.2/147.1；

Impact energy=30；

Parent ion fragmentation voltage=140；

Demethyl dextromethorphan Q1/Q3:258.2-157.1；258.2-133.1；

Impact energy=35；

Parent ion fragmentation voltage=135.

Bufuralol testing conditions:

Chromatographic condition:

Column temperature: 30 DEG C

Chromatographic column: ZORBAX SB-C18 (Agilent, 5 μm, 4.6 × 150mm)

Sampling volume: 1 μ L

Flow velocity: 0.6mL/min

Operation time: 10min

Table 4: bufuralol mobile phase ratio

Time (min)	10mM ammonium acetate (%, v/v)	Methanol (%, v/v)
			0.01	30	70
0.8	5	95
			3	5	95
4.01	30	70
			10	30	70

Mass Spectrometry Conditions:

Ionogenic temperature (TEM): 350 DEG C；

Curtain gas speed: 11L/min；

Capillary voltage: 4000V；

Bufuralol Q1/Q3:262.2/188.1；268.2/186.1；

Impact energy=10；

Parent ion fragmentation voltage=110；

1 '-hydroxyl bufuralol Q1/Q3:278.2/186.1；278.2/159.1；

Impact energy=15；

Parent ion fragmentation voltage=90.

4) LC-MS/MS detects data analysis

The different dextromethorphan of gradient concentration of preparation, bufuralol and corresponding metabolite demethyl dextromethorphan, 1 '-hydroxyl Base bufuralol (dextromethorphan: 2500,1250,500,250,100,20ng/mL；1500,750 demethyl dextromethorphan:, 300、150、50、25ng/mL；Bufuralol: 1000,500,250,100,50,25ng/mL；1 '-hydroxyl bufuralol: 800, 400,200,100,50,25ng/mL), generate standard curve after utilizing LC-MS detection, and in order to detect various CYP2D6 albumen To dextromethorphan and the metabolic condition of bufuralol, then represent the metabolism of this probe medicine with [product/(product+substrate)] Rate, after internal reference Gluc data calibration, represents saltant type egg with the ratio of the metabolic rate of saltant type Yu the metabolic rate of wild type White enzymatic activity.Every kind of albumen, for In vitro metabolism experiment repetition 3-4 time respectively of each probe medicament, is added up after averaging Result is shown in Table 5 respectively, table 6 and Fig. 3；The typical Mass Spectrometer Method result of dextromethorphan and bufuralol is shown in Fig. 4, figure by various albumen 5.The metabolic capacity of CYP2D6 variant is the strongest, and the peak area at substrate (dextromethorphan or bufuralol) peak will be the least, and produces The peak area at thing (demethyl dextromethorphan or 1 '-hydroxyl bufuralol) peak will be the biggest, it can be seen that new variant The metabolic capacity of V327M substantially relatively wild type reduces, again smaller than typical case mutant CYP2D6.2, but more than typical case's mutant CYP2D6.10。

The enzymatic activity result of table 5:293FT cell incubation probe medicament dextromethorphan

* refer to and the relative value of wild type CYP2D6.1

* p value < 0.05

The enzymatic activity result of table 6:293FT cell incubation probe medicament bufuralol

* refer to and the relative value of wild type CYP2D6.1

* p value < 0.05

Testing result shows, relative to wild type CYP2D6.1 type, it is known that typical defect type mutant CYP2D6.2 is to the right side The metabolic activity fall about about 13% of dromethan, to the range of decrease under the metabolic activity of another probe medicament bufuralol Degree is about 16%；Another typical defect type mutant CYP2D6.10 for two kinds of probe medicaments metabolic activity all substantially under Fall (fall is about 90%), this result is basically identical with existing document, shows the number that our vitro detection system obtains According to having the highest credibility (seeing list of references 6,7).

Utilize this vitro detection system it can be seen that V327M mutant is to probe medicament dextromethorphan and bufuralol Metabolic activity is respectively the 64.05% and 65.26% of wild type.Statistical analysis shows, compared with wild type, and V327M mutant Metabolic activity decline, there is significant difference, the metabolic activity pointing out this sudden change can cause expressed enzyme substantially reduces.Cause This, in practice, need to consider suitably to regulate the individuality carrying this genotype on dosage, as reduced medicine Usage amount and the generation avoiding adverse effect.This medicine by gene targeting adjusts the medicine bigger for individual variation Thing (such as imipramine, amitriptyline etc.) is even more important.

List of references

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 spoils, and Gong Sen, Ji Hongyan wait .CYP2D6 gene pleiomorphism and clinical meaning thereof. medicine 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.Pharmacogenetics 2001,11:417-427.

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: allelic sequences

ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCG CCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCC AGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGCAGAGGTGCTGAGGCTCCCCTACCA GAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAGAAGCCAGGCTGAGAAGGGGAAGCAGGTTTGGGGGA CGTCCTGGAGAAGGGCATTTATACATGGCATGAAGGACTGGATTTTCCAAAGGCCAAGGAAGAGTAGGGCAAGGGCC TGGAGGTGGAGCTGGACTTGGCAGTGGGCATGCAAGCCCATTGGGCAACATATGTTATGGAGTACAAAGTCCCTTCT GCTGACACCAGAAGGAAAGGCCTTGGGAATGGAAGATGAGTTAGTCCTGAGTGCCGTTTAAATCACGAAATCGAGGA TGAAGGGGGTGCAGTGACCCGGTTCAAACCTTTTGCACTGTGGGTCCTCGGGCCTCACTGCCTCACCGGCATGGACC ATCATCTGGGAATGGGATGCTAACTGGGGCCTCTCGGCAATTTTGGTGACTCTTGCAAGGTCATACCTGGGTGACGC ATCCAAACTGAGTTCCTCCATCACAGAAGGTGTGACCCCCACCCCCGCCCCACGATCAGGAGGCTGGGTCTCCTCCT TCCACCTGCTCACTCCTGGTAGCCCCGGGGGTCGTCCAAGGTTCAAATAGGACTAGGACCTGTAGTCTGGGGTGATC CTGGCTTGACAAGAGGCCCTGACCCTCCCTCTGCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGC CTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCG ACCGCCCGCCTGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGCAAGCAGCGGTGGGGACAGAGA CAGATTTCCGTGGGACCCGGGTGGGTGATGACCGTAGTCCGAGCTGGGCAGAGAGGGCGCGGGGTCGTGGACATGAA ACAGGCCAGCGAGTGGGGACAGCGGGCCAAGAAACCACCTGCACTAGGGAGGTGTGAGCATGGGGACGAGGGCGGGG CTTGTGACGAGTGGGCGGGGCCACTGCCGAGACCTGGCAGGAGCCCAATGGGTGAGCGTGGCGCATTTCCCAGCTGG AATCCGGTGTCGAAGTGGGGGCGGGGACCGCACCTGTGCTGTAAGCTCAGTGTGGGTGGCGCGGGGCCCGCGGGGTC TTCCCTGAGTGCAAAGGCGGTCAGGGTGGGCAGAGACGAGGTGGGGCAAAGCCTGCCCCAGCCAAGGGAGCAAGGTG GATGCACAAAGAGTGGGCCCTGTGACCAGCTGGACAGAGCCAGGGACTGCGGGAGACCAGGGGGAGCATAGGGTTGG AGTGGGTGGTGGATGGTGGGGCTAATGCCTTCATGGCCACGCGCACGTGCCCGTCCCACCCCCAGGGGTGTTCCTGG CGCGCTATGGGCCCGCGTGGCGCGAGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGGCCTGGGCAAGAAG TCGCTGGAGCAGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTCGCCAACCACTCCGGTGGGTGATGGGC AGAAGGGCACAAAGCGGGAACTGGGAAGGCGGGGGACGGGGAAGGCGACCCCTTACCCGCATCTCCCACCCCCAGGA CGCCCCTTTCGCCCCAACGGTCTCTTGGACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTT CGAGTACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGC GCGAGGTGCGGAGCGAGAGACCGAGGAGTCTCTGCAGGGCGA GCTCCCGAGAGGTGCCGGGGCTGGACTGGGGCCT CGGAAGAGCAGGATTTGCATAGATGGGTTTGGGAAAGGACATTCCAGGAGACCCCACTGTAAGAAGGGCCTGGAGGA GGAGGGGACATCTCAGACATGGTCGTGGGAGAGGTGTGCCCGGGTCAGGGGGCACCAGGAGAGGCCAAGGACTCTGT ACCTCCTATCCACGTCAGAGATTTCGATTTTAGGTTTCTCCTCTGGGCAAGGAGAGAGGGTGGAGGCTGGCACTTGG GGAGGGACTTGGTGAGGTCAGTGGTAAGGACAGGCAGGCCCTGGGTCTACCTGGAGATGGCTGGGGCCTGAGACTTG TCCAGGTGAACGCAGAGCACAGGAGGGATTGAGACCCCGTTCTGTCTGGTGTAGGTGCTGAATGCTGTCCCCGTCCT CCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGACCCAGCTGGATGAGCTGCTAA CTGAGCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAG GTGAGAGTGGCTGCCACGGTGGGGGGCAAGGGTGGTGGGTTGAGCGTCCCAGGAGGAATGAGGGGAGGCTGGGCAAA AGGTTGGACCAGTGCATCACCCGGCGAGCCGCATCTGGGCTGACAGGTGCAGAATTGGAGGTCATTTGGGGGCTACC CCGTTCTGTCCCGAGTATGCTCTCGGCCCTGCTCAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCT GCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCA TGATCCTACATCCGGATATGCAGCGTGAGCCCATCTGGGAAACAGTGCAGGGGCCGAGGGAGGAAGGGTACAGGCGG GGGCCCATGAACTTTGCTGGGACACCCGGGGCTCCAAGCACAGGCTTGACCAGGATCCTGTAAGCCTGACCTCCTCC AACATAGGAGGCAAGAAGGAGTGTCAGGGCCGGACCCCCTGGGTGCTGACCCATTGTGGGGACGCATGTCTGTCCAG GCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCC TACACCACTGCCGTGATTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCG TGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGTAGGCCTGGCGCCCTCCTCACCCCAGCTCAGCACCAGCACCT GGTGATAGCCCCAGCATGGCTACTGCCAGGTGGGCCCACTCTAGGAACCCTGGCCACCTAGTCCTCAATGCCACCAC ACTGACTGTCCCCACTTGGGTGGGGGGTCCAGAGTATAGGCAGGGCTGGCCTGTCCATCCAGAGCCCCCGTCTAGTG GGGAGACAAACCAGGACCTGCCAGAATGTTGGAGGACCCAACGCCTGCAGGGAGAGGGGGCAGTGTGGGTGCCTCTG AGAGGTGTGACTGCGCCCTGCTGTGGGGTCGGAGAGGGTACTGTGGAGCTTCTCGGGCGCAGGACTAGTTGACAGAG TCCAGCTGTGTGCCAGGCAGTGTGTGTCCCCCGTGTGTTTGGTGGCAGGGGTCCCAGCATCCTAGAGTCCAGTCCCC ACTCTCACCCTGCATCTCCTGCCCAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTC TGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCT GCCTTTCTCAGCAGGTGCCTGTGGGGAGCCCGGCTCCCTGTCCCCTTCCGTGGAGTCTTGCAGGGGTATCACCCAGG AGCCAGGCTCACTGACGCCCCTCCCCTCCCCACAGGCCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGC TCTTCCTCTTCTTCACCTCCCTG CTGCAGCACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCA TGGTGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAG

SEQ ID NO.2: coded sequence

ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCG CCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCC AGAACACACCATACTGCTTCGACCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACGCCG GTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCC TGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGC GCGAGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGGCCTGGGCAAGAAGTCGCTGGAGCAGTGGGTGACC GAGGAGGCCGCCTGCCTTTGTGCCGCCTTCGCCAACCACTCCGGACGCCCCTTTCGCCCCAACGGTCTCTTGGACAA AGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACGACGACCCTCGCTTCCTCAGGCTGC TGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCTGAATGCTGTCCCCGTCCTCCTG CATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGACCCAGCTGGATGAGCTGCTAACTGA GCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGCCA AGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTGCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTG ACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATATGCAGCGCCGTGTCCAACAGGA GATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGA TTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGTGACATCGAAGTACAG GGCTTCCGCATCCCTAAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGGAGAA GCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCT CAGCAGGCCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAG CACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGAGCCCATC CCCCTATGAGCTTTGTGCTGTGCCCCGCTAG

SEQ ID NO.3: protein sequence

MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYPPGPLPLPGLGNLLHVDFQNTPYCFDQLRRRFGDVFSLQLAWTP VVVLNGLAAVREALVTHGEDTADRPPVPITQILGFGPRSQGVFLARYGPAWREQRRFSVSTLRNLGLGKKSLEQWVT EEAACLCAAFANHSGRPFRPNGLLDKAVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLL HIPALAGKVLRFQKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDENLRI VVADLFSAGMVTTSTTLAWGLLLMILHPDMQRRVQQEIDDVIGQVRRPEMGDQAHMPYTTAVIHEVQRFGDIVPLGV THMTSRDIEVQGFRIPKGTTLITNLSSVLKDEAVWEKPFRFHPEHFLDAQGHFVKPEAFLPFSAGRRACLGEPLARM ELFLFFTSLLQHFSFSVPTGQPRPSHHGVFAFLVSPSPYELCAVPR

SEQ ID NO.14: nucleic acid fragment

CATCCGGATATGCAGCGTGA

SEQ ID NO.15: nucleic acid fragment

TCCTACATCCGGATATGCAGCGTGAGCCCA

SEQ ID NO.16: nucleic acid fragment

CATGATCCTACATCCGGATATGCAGCGTGAGCCCATCTGG

SEQ ID NO.17: nucleic acid fragment

CTGCTCATGATCCTACATCCGGATATGCAGCGTGAGCCCATCTGGGAAAC

SEQ ID NO.18: nucleic acid fragment

ATCCTACATCCGGATATGCAG

SEQ ID NO.19: oligonucleotide fragment

GGCTCACGCTGCATA

SEQ ID NO.20: oligonucleotide fragment

GATGGGCTCACGCTGCATAT

SEQ ID NO.21: oligonucleotide fragment

CCAGATGGGCTCACGCTGCATATC

SEQ ID NO.22: oligonucleotide fragment

CACGCTGCATATCCGGATGT

SEQ ID NO.23: oligonucleotide fragment

GGGCTCACGCTGCATATCCGGATGTAGGA。

Claims

1. nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 2943rd corresponding to SEQ ID NO.1, and is SEQ At least 10 continuous nucleotides in nucleotide sequence shown in ID NO.1 or its reverse complementary sequence, wherein the 2943rd Nucleotide is A；Or described nucleic acid fragment comprises the mutational site of the 979th corresponding to SEQ ID NO.2, and is SEQ ID At least 10 continuous nucleotides in nucleotide sequence shown in NO.2 or its reverse complementary sequence, the wherein nucleoside of the 979th Acid is A.

Nucleic acid fragment the most according to claim 1, it is characterised in that the length of described nucleic acid fragment is 10-100,101- 200,201-500 or 501-1000 nucleotide.

Nucleic acid fragment the most according to claim 1, it is characterised in that the length of described nucleic acid fragment be 10-20,21-30, 31-40,41-50,51-60,61-100 or 101-300 nucleotide.

Nucleic acid fragment the most according to claim 1, it is characterised in that described nucleic acid fragment is SEQ ID NO.1,2 or 14- Sequence shown in 18.

5. for detecting and/or analyze a test kit for single base mutation, including according to described in any one of claim 1-4 Nucleic acid fragment.

6. according to the answering in the medicine of preparation detection CYP2D6 gene mutation of the nucleic acid fragment described in any one of claim 1-4 With；Or the application in preparation is used as the check mark thing of detection CYP2D6 gene mutation.

7.CYP2D6 albumen, described protein sequence is the sequence shown in SEQ ID NO.3.