CN103614390B - Comprise the CYP2D6 gene fragment of 2519A > C sudden change, coded protein fragments and application thereof - Google Patents
Comprise the CYP2D6 gene fragment of 2519A > C sudden change, coded protein fragments and application thereof Download PDFInfo
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Abstract
The invention belongs to field of biology, relate to the single base mutation of the 2519th, CYP2D6 allelotrope, described site sports C by A.Particularly, the present invention relates to the application in the protein fragments of nucleic acid fragment and the corresponding encoded thereof comprising this mutational site, the reagent identifying described mutational site, detection method and this site, particularly identify the application of this site in direction of medication usage.
Description
Technical field
The invention belongs to field of biology, relate to the single base mutation of the 2519th, CYP2D6 allelotrope.More specifically, the present invention relates to the protein fragments of nucleic acid fragment and the corresponding encoded thereof comprising this mutational site, the reagent identifying described mutational site, detection method and the application of this site of qualification in direction of medication usage.
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, and exon 1 is positioned at 1620-5909 position).In human body, the amount of these cytopigment only accounts for 2% ~ 4% of liver enzyme total amount, but the metabolism of 20% ~ 30% medicine is clinically participated in, such medicine comprises thymoleptic, anti-arrhythmic, antipsychotic drug, anodyne, antitussive, antiemetic, antidiabetic drug and Bextra etc., studies its metabolic polymorphism and has very important clinical value (see reference 1).
CYP2D6 gene has height polymorphism.So far, included in NCBI snp database more than 300 mutational sites.Also existing more than 150, the allelotrope named by CYP450 Intemational Nomenclature committee member, also has multiple newfound saltant type not yet to be named (http://www.cypalleles.ki.se/cyp2d6.htm) in addition.Each allelotrope relates to point mutation, disappearance, insertion, rearrangement etc. in various degree, thus affects the activity of CYP2D6, and then the metabolic activity of impact to medicine, produces drug effect in various degree.Outside removing wild-type CYP2D6*1, current research is more and the saltant type that clinical meaning is larger mainly comprises following 7 kinds: CYP2D6*2, * 3, * 4, * 5, * 10, * 17, * 41, and wherein the widest, that most study, Chinese population research data are the relatively the abundantest saltant type of ethnic group distribution is CYP2D6*2(2850C>T; 4180G>C) and CYP2D6*10(100C>T; 4180G>C) (see reference 1,2,3).
According to current clinical studies show, this polymorphism of CYP2D6 gene causes the major cause of CYP2D6 enzymic activity significant difference between individuals, carry the huge 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, study CYP2D6 gene pleiomorphism and will provide important scientific basis (see reference 3,4) to clinical rational drug use to the impact of curative effect of medication.
Summary of the invention
The object of this invention is to provide the new single base mutation site of CYP2D6 gene, comprise the nucleic acid fragment in this mutational site, its protein fragments of encoding 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 2519th corresponding to SEQ IDNO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, wherein the Nucleotide of the 2519th is C; Or described nucleic acid fragment comprises the mutational site of the 745th 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 745th is C; Or described nucleic acid fragment is the reverse complementary sequence of above-mentioned nucleic acid fragment.
Second aspect of the present invention be to provide with containing corresponding to the 2519th of SEQ ID NO.1 or correspond to the allelotrope fragment in the mutational site of the 745th or the allele specific oligonucleotide of all or part of hybridization of its reverse complementary sequence of SEQ ID NO.2, wherein the Nucleotide in the mutational site of the 2519th of SEQ ID NO.1 or the 745th of SEQ ID NO.2 is C; Described allelotrope fragment is at least 10 continuous nucleotides in SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.2 or its reverse complementary sequence.
3rd aspect of the present invention is to provide the test kit for detecting and/or analyze single base mutation of the present invention, described test kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprises the nucleic acid fragment that can comprise this single base as single base mutation described in primer amplification but not; Described single base corresponds to the 2519th of SEQ ID NO.1 or the 745th of SEQ ID NO.2.
4th aspect of the present invention is to provide nucleic acid fragment of the present invention or oligonucleotide is detecting the application in CYP2D6 transgenation, and wherein said nucleic acid fragment or oligonucleotide are used as probe or primer; Or nucleic acid fragment of the present invention or oligonucleotide are for the preparation of the application of medicine detecting CYP2D6 transgenation; Or nucleic acid fragment of the present invention or oligonucleotide are used as the application of the check mark thing detecting CYP2D6 transgenation.
5th aspect of the present invention is to provide a kind of medication guide, comprises the single base mutation corresponding to the 2519th of SEQ ID NO.1 or the 745th of SEQ ID NO.2 detecting CYP2D6 gene in testing sample; According to the sudden change detected, adjust by the dosage of the medicine of CYP2D6 metabolism.
6th aspect of the present invention is to provide the method for analysis of nucleic acids, and described method comprises in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.1 analyzed in testing sample the Nucleotide that corresponds to the 2519th or analyzes in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.2 in testing sample the Nucleotide corresponding to the 745th.
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 proline(Pro) of the 249th 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 encoding sequence that comprise new single base mutation.The 745th Nucleotide that this gene is corresponding to SEQ ID NO.2 sports C(745A>C by A), thus the amino acid causing it to encode sports proline(Pro) by Threonine, namely corresponds to the proline(Pro) of the 249th of SEQ ID NO.3.The CYP2D6 albumen (called after T249P) of this sudden change is to metabolic activity reduction compared with wild-type of medicine.The medication of this single base mutation to the individuality carrying this mutational site has directive significance.
Accompanying drawing explanation
Fig. 1 is the heterozygous carriers's Sequencing chromatogram corresponding to SEQ ID NO.1 sequence of the present invention in embodiment 1, and wherein arrow indication is CYP2D6 gene the 2519th Nucleotide;
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(deficient mutants) and T249P albumen of the present invention for the metabolic capacity detected result of Dextromethorphane Hbr and bufuralol, wherein * represents p value <0.05;
Fig. 4 is the CYP2D6.1(wild-type shown in embodiment 2), CYP2D6.2(deficient mutants), CYP2D6.10(deficient mutants) and T249P 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 substrates dextromethorphan (right side) and meta-bolites demethyl Dextromethorphane Hbr (left side) thereof; Wherein X-coordinate represents 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;
Fig. 5 is the CYP2D6.1(wild-type shown in embodiment 2), CYP2D6.2(deficient mutants), CYP2D6.10(deficient mutants) and T249P albumen of the present invention detect collection of illustrative plates for the typical LC-MS/MS of bufuralol metabolism; Arrow indication place is respectively the fignal center of substrate bufuralol (right side) and meta-bolites 1 '-hydroxyl bufuralol (left side) thereof; Wherein X-coordinate represents retention time, and the retention time of bufuralol and 1 '-hydroxyl bufuralol is respectively 6.9 and 5.1 minutes, ordinate zou expression signal response value cps.
Embodiment
By following embodiment, the present invention is described, but content of the present invention is not limited thereto.
As illustrated without other, " nucleic acid fragment " of the present invention is made up of Nucleotide or its analogue, can be the fragment of DNA, RNA or its analogue; Can be strand or double-strand; Can be natural (as genomic) or synthesis.
In the present invention, " sudden change " refers to the gene detected, and namely there is the nucleotide site different from wild-type CYP2D6 gene order in CYP2D6 gene." mutational site " refers to the position that base is undergone mutation.In the present invention, described mutational site corresponds to the 745th in sequence shown in the 2519th of sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
International P450 allelotrope NK specifies about in CYP2D6 allelotrope naming rule: in CYP2D6 genomic dna reference sequences (GenBank number of registration M33388), first base A of initiator codon ATG is as allelic 1st of CYP2D6 (first base A of initiator codon is positioned at the 1620th of M33388 sequence), then the catastrophe point that the present invention determines is positioned at allelic 2519th 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 performance in encoding sequence (i.e. CDS).Those skilled in the art, according to detected sample, can detect this mutational site on genomic dna or mRNA level in-site.In the application, SEQ ID NO.1 is the CYP2D6 allelic sequences of the present invention of the stipulative definition according to international P450 allelotrope NK, and wherein the 2519th is the mutational site that the present invention relates to.SEQ ID NO.2 is the cDNA sequence of the CYP2D6 gene with described mutational site, and wherein the 745th is the mutational site that the present invention relates to.Those skilled in the art are known, and in this article, the 2519th site corresponding to SEQ ID NO.1 and the 745th the site synonym corresponding to SEQ ID NO.2 are used mutually.
In the present invention, " allele-specific " refers to hybridize with allelotrope specifically, as hybridized under high stringency conditions, makes to identify that the 745th Nucleotide corresponding to sequence shown in the 2519th of sequence shown in SEQ ID NO.1 or SEQ IDNO.2 is C.
In the present invention, Nucleotide and amino acid whose abbreviation adopt abbreviation mode well known in the art, and as in Nucleotide, A represents VITAMIN B4, G represents guanine, and C represents cytosine(Cyt), and T represents thymus pyrimidine.In amino acid, A represents L-Ala, and R represents arginine, and N represents l-asparagine, D represents aspartic acid, and C represents halfcystine, and Q represents glutamine, and E represents L-glutamic acid, G represents glycine, and H represents Histidine, and I represents Isoleucine, and L represents leucine, K represents Methionin, and M represents methionine(Met), and F represents phenylalanine, P represents proline(Pro), and S represents Serine, and T represents Threonine, W represents tryptophane, and Y represents tyrosine, and V represents α-amino-isovaleric acid.
Content of the present invention is the new single base mutation site based on CYP2D6 gene.Described mutational site is the coding region being positioned at CYP2D6 gene, and corresponding to the 745th of SEQ ID NO.2, this site sports C by the A of wild-type; In addition, proline(Pro) (T249P) is sported by the 249th of albumen of the CYP2D6 genes encoding of this sudden change by Threonine.
In first, the invention provides nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 2519th corresponding to SEQ IDNO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, and wherein the Nucleotide of the 2519th is C; Or described nucleic acid fragment comprises the mutational site of the 745th 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 745th is C; Or be the reverse complementary sequence of above-mentioned nucleic acid fragment.
In one embodiment, the length of described nucleic acid fragment can be as 10-100,101-200,201-500 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 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.
Second aspect of the present invention be to provide with containing corresponding to the 2519th of SEQ ID NO.1 or correspond to the allelotrope fragment in the mutational site of the 745th or the allele specific oligonucleotide of all or part of hybridization of its reverse complementary sequence of SEQ ID NO.2, wherein the Nucleotide in the mutational site of the 2519th of SEQ ID NO.1 or the 745th of SEQ ID NO.2 is C; Described allelotrope fragment is at least 10 continuous nucleotides in SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.2 or its reverse complementary sequence.
In one embodiment, described oligonucleotide is used as probe.Described probe can under high stringency conditions with comprise the target sequence specific hybrid in mutational site.It is known to those skilled in the art that described probe does not need and target sequence complete complementary, if can with target sequence specific hybridization.In preferred embodiments, described hybridization conditions can meet make probe only with 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 the center of probe sequence or about center.
In another embodiment, the described oligonucleotide primer of the DNA synthesis that coaches, sequencing primer as known in the art or synthetic primer etc.Described primer does not need and template complete complementary, but should synthesize to instruct DNA with template Complementary hybridization.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, 3rd aspect of the present invention is to provide for detecting 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 comprises the nucleic acid fragment that can comprise this single base as single base mutation described in primer amplification but not; Described single base corresponds to the 2519th of SEQ ID NO.1 or the 745th of SEQ ID NO.2.Preferably, described test kit comprises SEQ ID NO.6 and/or SEQ ID NO.7 and/or the sequence fragment shown in SEQ ID NO.11.
4th aspect of the present invention is to provide nucleic acid fragment of the present invention or oligonucleotide for detecting the application of CYP2D6 transgenation, and wherein said nucleic acid fragment or oligonucleotide are used as probe or primer; Or nucleic acid fragment of the present invention or oligonucleotide are for the preparation of the application of medicine detecting CYP2D6 transgenation; Or nucleic acid fragment of the present invention or oligonucleotide are used as the application of the check mark thing detecting CYP2D6 transgenation.
5th aspect of the present invention is to provide medication guide, comprises the base corresponding to the 2519th of SEQ ID NO.1 or the 745th of SEQ ID NO.2 detecting CYP2D6 gene in testing sample.When the CYP2D6 gene detected is when the site corresponding to the 2519th of SEQ ID NO.1 or the 745th of SEQ ID NO.2 is C, adjust the dosage of the medicine through CYP2D6 metabolism accordingly.In the particular embodiment, when CYP2D6 gene is when the site of the 745th of SEQ ID NO.2 is C, the CYP2D6 protease activity of this genes encoding declines, therefore needs adjustment through the dosage of the medicine of CYP2D6 metabolism, assigns dose as follows.
The medicine through 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.
6th aspect of the present invention is to provide the method for analysis of nucleic acids, and described method comprises in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.1 analyzed in testing sample the Nucleotide that corresponds to the 2519th or analyzes in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.2 in testing sample the Nucleotide corresponding to the 745th.
In one embodiment, described method can be restriction fragment length polymorphism analysis (RFLP).Whether those skilled in the art can according to content design experiment of the present invention analyze the Nucleotide of the 745th in the nucleic acid of the sequence of the Nucleotide of the 2519th in the nucleic acid of the sequence of SEQ ID NO.1 or SEQ ID NO.2 for C.
In another embodiment, described method can be sequencing, comprise and being separated and the nucleotide sequence measured from genomic dna or RNA, analyze and wherein comprise corresponding to corresponding to the Nucleotide of the 2519th or comprising corresponding to corresponding to whether the Nucleotide of the 745th is C in the nucleic acid of the sequence of SEQ ID NO.2 in the nucleic acid of the sequence of SEQ ID NO.1.Sequencing can be any available sequence measurement known in the art.Sequencing primer can design according to the general knowledge of those skilled in the art, as the upstream and downstream appropriate position design primer in site to be detected, with the fragment of expanding packet containing this site to be measured, thus judges the Nucleotide in this site.Also oligonucleotide of the present invention can be adopted as primer sequence.
In another embodiment, described method is the method utilizing probe hybridization, and whether the Nucleotide comprised in identification and detection sample specifically corresponding to corresponding to the 745th in the Nucleotide corresponding to the 2519th in the nucleic acid of the sequence of SEQ ID NO.1 or the nucleic acid comprising corresponding to the sequence of SEQ ID NO.2 is C; The probe adopted in described method is oligonucleotide of the present invention.Such as, from testing sample, isolate nucleic acid, under the condition allowing probe and the specific target sequence that may exist in nucleic acid to hybridize, probe is contacted with nucleic acid; The hybridization that can be detected can be realized by the probe that detectable reagent is labeled by using; Such as, form the enzyme that can detect product with radio isotope, fluorescence dye or energy catalysis and carry out label probe.Label probe, to detect in sample with label probe the method that whether there is target sequence be all well-known to those skilled in the art.
In a kind of concrete embodiment, the method detecting the Nucleotide of the 2519th corresponding to SEQ ID NO.1 is provided, comprises with Taqman probe SNP detection method:
1) design primer to be used for specific amplification and to comprise the PCR primer of the 2519th corresponding to SEQ ID NO.1, design two Taqman-MGB probes, respectively for A and the C allelotrope of the 2519th corresponding to SEQ ID NO.1 simultaneously.
Design of primers principle is:
(1) choose should at the conservative section of gene for sequence;
(2) avoid primer self or and primer between form more than 4 or 4 and match continuously, avoid primer self to form pili annulati card structure;
(3) primer length is at 18 to 24 Nucleotide;
(4) Tm value at 55-65 DEG C, GC content at 40%-60%;
(5) the Tm value difference between primer avoids exceeding 2 DEG C;
(6) 3 ' end of primer is avoided using base A, and 3 ' of primer holds the base avoiding appearance more than 3 or 3 consecutive identical;
(7) pcr amplified fragment length is at 50bp-150bp;
(8) last 5 Nucleotide of prime end can not have G and C more than 2.
Taqman MGB probe design principle is:
(1) 5 ' end of probe is avoided occurring G;
(2) Tm value should be 65-67 DEG C;
(3) shorten Taqman MGB probe, but probe length is no less than 13bp as far as possible;
(4) avoid the base duplicated, especially G base as far as possible, avoid the G of appearance more than 4 or 4 to repeat;
(5) mutational site of probe is placed on as far as possible the place of middle 1/3.
Fluorophor can adopt FAM, VIC etc. to mark two allelotrope.
2) utilize above-mentioned primer and probe, real-time quantitative PCR is carried out to sample to be tested.
PCR condition: 95 DEG C of denaturations enter 30 amplification cycles after 10 minutes: 92 DEG C of sex change 12 seconds, 60 DEG C of annealing and extend 1 minute (this stage detects fluorescent signal).
3) data analysis.
According to the power of sample two kinds of fluorescence, analysis design mothod result, judges whether sample to be tested CYP2D6 gene exists 2519A>C sudden change.
In the present invention, described sample can be any sample comprising nucleic acid, as blood; Preferred described sample comes from people.Described nucleic acid can be DNA or coding RNA, is preferably genomic dna.The method of analysis of nucleic acids of the present invention can with DNA or RNA for target compound.Those skilled in the art are known, when taking DNA as detection target compound, analyze in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.1 in testing sample the Nucleotide corresponding to the 2519th, the probe used or primer are according to the sequences Design of SEQ ID NO.1; When being when detecting target compound with RNA, analyze in the nucleic acid comprising corresponding to the sequence of SEQID NO.2 in testing sample the Nucleotide corresponding to the 745th, the probe used or primer are according to the sequences Design of SEQ ID NO.2.
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 proline(Pro) of the 249th 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.
To further illustrate the present invention by specific embodiment below, but following specific embodiment is only for exemplary object.
Embodiment 1: the qualification 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, design sequencing primer carries out sequence amplification, order-checking to 9 of CYP2D6 gene exons, analyzes its CYP2D6 gene and whether there is mutational site.
1) DNA is extracted:
5ml vein EDTA anticoagulated blood sample is taked from measured; Then according to common salting-out process and/or adopt special DNA extraction kit (DNA extraction kit of purchased from American Omega company) to extract the genomic dna of blood sample to be measured.
2) pcr amplification:
Design of amplification primers, increases 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: the genomic dna of 1 × GC PCR damping fluid, 1.5mM MgCl2,100ng, upstream and downstream primer are 0.2 μM, dNTP is the LATaqDNA polysaccharase 1.5U of 0.2mM, TaKaRa company.Use the GeneAmp PCR System9700 amplification instrument amplification of American AB I company.Pcr amplification loop parameter is as follows: 94 DEG C of denaturations 2 minutes, 94 DEG C of sex change 30 seconds, and 60 DEG C of annealing 30 seconds, 68 DEG C extend 3 minutes, extend 3 minutes again after 35 circulations.Primer sequence information is in table 1.
Table 1: amplimer is to sequence information
3) purifying amplified production:
By the amplified production of acquisition according to MultiScreen HTS
tMthe operation instruction of test kit (Millipore company of the U.S.), the DNA carrying out object band reclaims purifying.
4) check order:
With the product after recovery for template, use sequencing primer to carry out order-checking PCR according to BigDye Terminator v3.1 sequencing kit (American AB I company) operation instruction to react, reaction terminates rear purifying amplified production, uses the Prism3730XL type gene sequencer of American AB I company to carry out being separated the sequence with interpretation amplified production.Sequencing primer sequence information is 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 and wild-type CYP2D6*1 sequence (GenBank number of registration M33388) are compared.
By compare of analysis, find have the CYP2D6 genomic dna sequence of 1 people to carry a kind of brand-new mutation type in 2129 routine experimenters, the Nucleotide of allelic 2519th corresponding of CYP2D6 becomes C(as shown in Figure 1 from A, and wherein M represents A and C heterozygosis).This sudden change is positioned at the 5th exon of CYP2D6 gene, and catastrophe point is positioned at cDNA the 745th, infers in the protein of this CYP2D6 genes encoding accordingly, and the 249th amino acids sports proline(Pro) (P) by Threonine (T).This sudden change now by P450 NK called after neomorph CYP2D6*93, but is not yet externally announced.
The present embodiment exemplarily gives the method in qualification new mutant site.Those skilled in the art clearly can learn the method detecting specifically and comprise the 2519th Nucleotide corresponding to SEQ ID NO.1 in testing sample according to foregoing: the nucleic acid in sample separation, 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 IDNO.11, the product of amplification is checked order; By sequencing result and wild-type results comparison, analyze the Nucleotide in the 2519th site corresponding to SEQ ID NO.1.
Embodiment 2: vitro enzyme metabolic activity is analyzed
According to existing result of study, wild-type (the * 1 type) metabolic activity to various medicine is all higher, 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 lower than * 2 types (see document 7,8).Therefore, existing one is like this known together in the art: the enzyme expressed by same genotype can represent the metabolic activity to other substrate medicine to the metabolic activity of specific substrate.Thus, enzyme expressed by a certain genotype can analogize the metabolic activity (e.g., the metabolic activity of the enzyme metabolic activity of enzyme this genotype expressed by and wild-type expressed by can be compared) of the enzyme expressed by this genotype to other substrate medicine to specific substrate metabolic activity data.
In the present embodiment, according to above-mentioned mutational site, with wild-type CYP2D6(*1) gene is template, the rite-directed mutagenesis Nucleotide (becoming C from A) of the 745th of CYP2D6 gene coding region, the expression vector of construction expression saltant type CYP2D6 albumen (called after T249P or CYP2D6.93), after transfection 293FT cell, add CYP2D6 Specific probe---Dextromethorphane Hbr and bufuralol, after hatching, the In vitro metabolism being detected this saltant type CYP2D6 albumen by analysis substrate and meta-bolites amount is active, to judge compared with wild-type CYP2D6.1, whether its enzymatic metabolism activity changes.This experimental design is similar to us at CYP2C9 new mutant medicine in-vitro for operation steps during activation analysis, and its science is approved (see reference document 9) by the magazine Pharmacogenomics J that International Pharmaceutical genomics field is the most authoritative.
1) vivoexpression of CYP2D6 varient
To comprise wild-type CYP2D6(*1) plasmid vector (buying from ThermoScientific company) of full-length cDNA is template, utilizes side-directed mutagenesis to obtain the cDNA of CYP2D6*2, CYP2D6*10 and T249P mutant of the present invention respectively.Side-directed mutagenesis is techniques well known, and those skilled in the art, according to the template determined and target, unambiguously can know how to complete this step.By each goal gene correct for sequence after testing and reference gene Gluc(secretor type luciferase, its translation product can be secreted in substratum, and fluorescent signal detected by particular agent box; Its skeleton carrier is pIRES pGluc-Basic, purchased from NEB company, article No. N8082S) be connected to double gene expression vector pIRES(respectively purchased from Clontech company, article No. 631605) A, B multiple clone site in, under making a kind of goal gene and reference gene be positioned at the control of same CMV promoter, Fig. 2 is shown in by the final double gene expression vector pIRES-Gluc-2D6(structural representation collection of illustrative plates that obtains).Build and comprise CYP2D6(*1 respectively) four kinds of CYP2D6 double gene expression vectors of the cDNA of cDNA, CYP2D6*2cDNA, CYP2D6*10cDNA and T249P mutant of the present invention.
By 5 × 10
5individual 293FT cell (people's renal epithelial cell is originated, purchased from Invitrogen company) is evenly 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 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 cultivation after 24 hours, resuspendedly (use EP pipe to carry out) in 300 μ l substratum single hole cell dissociation in 6 orifice plates.Use classical probe medicine Dextromethorphane Hbr (buying from Sigma Co., USA) of two kinds of CYP2D6 and bufuralol (buying from Canadian Toronto Research Chemicals company) to detect the activity of this enzyme, the final concentration of two kinds of medicines is respectively 20 μMs and 10 μMs.At 37 DEG C, at 5%CO
2in incubator, 3h is hatched in 300rpm concussion.From CO
2culture is taken out in incubator, add the 0.1MNaOH of 20 μ L and to shake vortex 1min(reason be bufuralol and Dextromethorphane Hbr is all weak base, solution NaOH will exist with molecular conformation after alkalizing, and easily enter organic phase thus be extracted out in next step extraction process).Add 800 μ L glacial acetic acid ethyl esters, concussion vortex 2min is placed on-40 DEG C of refrigerator 30min, until lower floor is freezing.Take out sample, at 4 DEG C, with the centrifugal 10min of 12000g.Upper strata ethyl acetate is transferred in new EP pipe, ethyl acetate dries up by 37 DEG C of Nitrogen evaporators.Add 200 μ L initial flow phase solution redissolution separately, with the centrifugal 5min of 12000g after concussion vortex 1min.Supernatant uses initial flow phase solution to be transferred in sample injection bottle by after 1:10 dilution.
3) LC-MS/MS detects
After abstraction purification, sample carries out LC-MS/MS detection on 1260-6410 type instrument (Agilent company of the U.S.), and HPLC adopts ZORBAX SB-C18 post (150mm × 4.6mm, diameter 5 μm).
Dextromethorphane Hbr 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
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 |
Mass Spectrometry Conditions:
Electron spray(ES) (ESI) ion source (positive ion), many reaction detection scanning (MRM) patterns;
Ionogenic temperature (TEM): 300 DEG C;
Curtain gas speed: 11L/min;
Capillary voltage: 4000V;
Dextromethorphane Hbr Q1/Q3:272.2/213.1; 272.2/147.1;
Impact energy=30;
Cracked voltage=140 of parent ion;
Demethyl Dextromethorphane Hbr Q1/Q3:258.2-157.1; 258.2-133.1;
Impact energy=35;
Cracked voltage=135 of parent ion.
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
Working time: 10min
Table 4: bufuralol mobile phase ratio
| Time (min) | 10mM ammonium acetate (%, v/v) | Methyl alcohol (%, v/v) |
| 0.01 | 30 | 70 |
| 0.8 | 5 | 95 |
| 3 | 5 | 95 |
| 4.01 | 30 | 70 |
| 10 | 30 | 70 |
Mass Spectrometry Conditions:
Electron spray(ES) (ESI) ion source (positive ion), many reaction detection scanning (MRM) patterns;
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;
Cracked voltage=110 of parent ion;
1 '-hydroxyl bufuralol Q1/Q3:278.2/186.1; 278.2/159.1;
Impact energy=15;
Cracked voltage=90 of parent ion.
4) LC-MS/MS detects data analysis
Prepare the Dextromethorphane Hbr of different gradient concentration, bufuralol and corresponding meta-bolites demethyl Dextromethorphane Hbr, 1 '-hydroxyl bufuralol (Dextromethorphane Hbr: 2500,1250,500,250,100,20ng/mL; Demethyl Dextromethorphane Hbr: 1500,750,300,150,50,25ng/mL; Bufuralol: 1000,500,250,100,50,25ng/mL; 1 '-hydroxyl bufuralol: 800,400,200,100,50,25ng/mL), typical curve is generated after utilizing LC-MS to detect, and in order to detect the metabolic condition of various CYP2D6 albumen to Dextromethorphane Hbr and bufuralol, then the metabolic rate of this probe medicine is represented with [product/(product+substrate)], after internal reference Gluc data calibration, represent the enzymic activity of mutein with the ratio of the metabolic rate of saltant type and the metabolic rate of wild-type.Often kind of albumen repeats 4 times respectively for the experiment of different pharmaceutical, and after averaging, statistics is respectively in table 5, table 6 and Fig. 3; The typical mass spectrometric detection of various albumen to Dextromethorphane Hbr and bufuralol the results are shown in Figure 4, Fig. 5.The metabolic capacity of CYP2D6 varient is stronger, the peak area at substrate (Dextromethorphane Hbr or bufuralol) peak will be less, and the peak area at product (demethyl Dextromethorphane Hbr or 1 '-hydroxyl bufuralol) peak will be larger, as can be seen from the figure, the metabolic capacity obviously comparatively wild-type reduction of new varient CYP2D6.93, and be less than typical mutant CYP2D6.2 and CYP2D6.10.
The enzymic activity result of table 5:293FT cell incubation probe medicament Dextromethorphane Hbr
* the relative value with wild-type CYP2D6.1 is referred to
* p value <0.05
The enzymic activity result of table 6:293FT cell incubation probe medicament bufuralol
* the relative value with wild-type CYP2D6.1 is referred to
* p value <0.05
Detected result shows, relative to wild-type CYP2D6.1 type, the metabolic activity of known typical defect type mutant CYP2D6.2 to Dextromethorphane Hbr declines not obvious (about 5%), but obviously declines to the metabolic activity of another probe medicament bufuralol, and fall is about 16%; Another typical defect type mutant CYP2D6.10 all obviously declines (amplitude that reduces is higher than 90%) for the metabolic activity of two kinds of probe medicaments, this result and existing document basically identical, show we vitro detection system obtain data there is very high confidence level (see reference 7,8).
This vitro detection system is utilized to find out: the metabolic activity of T249P mutant to probe medicament Dextromethorphane Hbr and bufuralol is respectively 9.19% and 1.51% of wild-type.Statistical analysis shows, compared with wild-type, the metabolic activity of T249P mutant declines, and has significant difference, points out this sudden change that the metabolic activity of expressed enzyme can be caused obviously to reduce.Therefore, in practice, need to consider suitably to regulate on dosage carrying this genotypic individuality, as reduced the usage quantity of medicine and avoiding the generation of adverse drug reaction.This adjustment of the medicine by gene targeting is even more important for the medicine (as imipramine, amitriptyline etc.) that individual difference is larger.
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 spoils, and Gong Sen, Ji Hongyan, wait .CYP2D6 gene pleiomorphism and clinical meaning thereof. medical Leader .2012,31 (10): 1337-1340.
5.Hersberger M,Marti-Jaun J,Rentsch K,Hanseler E.Rapid detection of the CYP2D6*3,CYP2D6*4,and CYP2D6*6alleles by tetra-primer PCR and of the CYP2D6*5allele by multiplex long PCR.Clin Chem.2000,46:1072-1077.
6.Lovlie R,Daly AK,Molven A,Idle JR,Steen VM.Ultrarapid metabolizers of debrisoquine:characterization and PCR-based detection of alleles with duplication of the CYP2D6gene.FEBS Lett.1996,392:30-34.
7.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.
8.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.
9.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.
Sequence:
SEQ ID NO.1: allelic sequences
ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGCAGAGGTGCTGAGGCTCCCCTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAGAAGCCAGGCTGAGAAGGGGAAGCAGGTTTGGGGGACGTCCTGGAGAAGGGCATTTATACATGGCATGAAGGACTGGATTTTCCAAAGGCCAAGGAAGAGTAGGGCAAGGGCCTGGAGGTGGAGCTGGACTTGGCAGTGGGCATGCAAGCCCATTGGGCAACATATGTTATGGAGTACAAAGTCCCTTCTGCTGACACCAGAAGGAAAGGCCTTGGGAATGGAAGATGAGTTAGTCCTGAGTGCCGTTTAAATCACGAAATCGAGGATGAAGGGGGTGCAGTGACCCGGTTCAAACCTTTTGCACTGTGGGTCCTCGGGCCTCACTGCCTCACCGGCATGGACCATCATCTGGGAATGGGATGCTAACTGGGGCCTCTCGGCAATTTTGGTGACTCTTGCAAGGTCATACCTGGGTGACGCATCCAAACTGAGTTCCTCCATCACAGAAGGTGTGACCCCCACCCCCGCCCCACGATCAGGAGGCTGGGTCTCCTCCTTCCACCTGCTCACTCCTGGTAGCCCCGGGGGTCGTCCAAGGTTCAAATAGGACTAGGACCTGTAGTCTGGGGTGATCCTGGCTTGACAAGAGGCCCTGACCCTCCCTCTGCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCCTGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGCAAGCAGCGGTGGGGACAGAGACAGATTTCCGTGGGACCCGGGTGGGTGATGACCGTAGTCCGAGCTGGGCAGAGAGGGCGCGGGGTCGTGGACATGAAACAGGCCAGCGAGTGGGGACAGCGGGCCAAGAAACCACCTGCACTAGGGAGGTGTGAGCATGGGGACGAGGGCGGGGCTTGTGACGAGTGGGCGGGGCCACTGCCGAGACCTGGCAGGAGCCCAATGGGTGAGCGTGGCGCATTTCCCAGCTGGAATCCGGTGTCGAAGTGGGGGCGGGGACCGCACCTGTGCTGTAAGCTCAGTGTGGGTGGCGCGGGGCCCGCGGGGTCTTCCCTGAGTGCAAAGGCGGTCAGGGTGGGCAGAGACGAGGTGGGGCAAAGCCTGCCCCAGCCAAGGGAGCAAGGTGGATGCACAAAGAGTGGGCCCTGTGACCAGCTGGACAGAGCCAGGGACTGCGGGAGACCAGGGGGAGCATAGGGTTGGAGTGGGTGGTGGATGGTGGGGCTAATGCCTTCATGGCCACGCGCACGTGCCCGTCCCACCCCCAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGCGCGAGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGGCCTGGGCAAGAAGTCGCTGGAGC AGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTCGCCAACCACTCCGGTGGGTGATGGGCAGAAGGGCACAAAGCGGGAACTGGGAAGGCGGGGGACGGGGAAGGCGACCCCTTACCCGCATCTCCCACCCCCAGGACGCCCCTTTCGCCCCAACGGTCTCTTGGACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCGGAGCGAGAGACCGAGGAGTCTCTGCAGGGCGAGCTCCCGAGAGGTGCCGGGGCTGGACTGGGGCCTCGGAAGAGCAGGATTTGCATAGATGGGTTTGGGAAAGGACATTCCAGGAGACCCCACTGTAAGAAGGGCCTGGAGGAGGAGGGGACATCTCAGACATGGTCGTGGGAGAGGTGTGCCCGGGTCAGGGGGCACCAGGAGAGGCCAAGGACTCTGTACCTCCTATCCACGTCAGAGATTTCGATTTTAGGTTTCTCCTCTGGGCAAGGAGAGAGGGTGGAGGCTGGCACTTGGGGAGGGACTTGGTGAGGTCAGTGGTAAGGACAGGCAGGCCCTGGGTCTACCTGGAGATGGCTGGGGCCTGAGACTTGTCCAGGTGAACGCAGAGCACAGGAGGGATTGAGACCCCGTTCTGTCTGGTGTAGGTGCTGAATGCTGTCCCCGTCCTCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGCCCCAGCTGGATGAGCTGCTAACTGAGCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGTGAGAGTGGCTGCCACGGTGGGGGGCAAGGGTGGTGGGTTGAGCGTCCCAGGAGGAATGAGGGGAGGCTGGGCAAAAGGTTGGACCAGTGCATCACCCGGCGAGCCGCATCTGGGCTGACAGGTGCAGAATTGGAGGTCATTTGGGGGCTACCCCGTTCTGTCCCGAGTATGCTCTCGGCCCTGCTCAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTGCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGTGAGCCCATCTGGGAAACAGTGCAGGGGCCGAGGGAGGAAGGGTACAGGCGGGGGCCCATGAACTTTGCTGGGACACCCGGGGCTCCAAGCACAGGCTTGACCAGGATCCTGTAAGCCTGACCTCCTCCAACATAGGAGGCAAGAAGGAGTGTCAGGGCCGGACCCCCTGGGTGCTGACCCATTGTGGGGACGCATGTCTGTCCAGGCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGATTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGTGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGTAGGCCTGGCGCCCTCCTCACCCCAGCTCAGCACCAGCACCTGGTGATAGCCCCAGCATGGCTACTGCCAGGTGGGCCCACTCTAGGAACCCTGGCCACCTAGTCCTCAATGCCACCACACTGACTGTCCCCACTTGGGTGGGGGGTCCAGAGTATAGGCAGGGCTGGCCTGTCCATCCAGAGCCCCCGTCTAGTGGGGAGACAAACCAGGACCTGCCAGAATGTTGGAGGACCCAACGCCTGCAGGGAGAGGGGGCAGTGTGGGTGCCTCTGAGAGGTGTGACTGCGCCCTGCTGTG GGGTCGGAGAGGGTACTGTGGAGCTTCTCGGGCGCAGGACTAGTTGACAGAGTCCAGCTGTGTGCCAGGCAGTGTGTGTCCCCCGTGTGTTTGGTGGCAGGGGTCCCAGCATCCTAGAGTCCAGTCCCCACTCTCACCCTGCATCTCCTGCCCAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCTCAGCAGGTGCCTGTGGGGAGCCCGGCTCCCTGTCCCCTTCCGTGGAGTCTTGCAGGGGTATCACCCAGGAGCCAGGCTCACTGACGCCCCTCCCCTCCCCACAGGCCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAGCACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAG
SEQ ID NO.2: encoding sequence
ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCCTGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGCGCGAGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGGCCTGGGCAAGAAGTCGCTGGAGCAGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTCGCCAACCACTCCGGACGCCCCTTTCGCCCCAACGGTCTCTTGGACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCTGAATGCTGTCCCCGTCCTCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGCCCCAGCTGGATGAGCTGCTAACTGAGCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTGCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGATTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGTGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCTCAGCAGG CCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAGCACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAG
SEQ ID NO.3: protein sequence
MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYPPGPLPLPGLGNLLHVDFQNTPYCFDQLRRRFGDVFSLQLAWTPVVVLNGLAAVREALVTHGEDTADRPPVPITQILGFGPRSQGVFLARYGPAWREQRRFSVSTLRNLGLGKKSLEQWVTEEAACLCAAFANHSGRPFRPNGLLDKAVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLLHIPALAGKVLRFQKAFLPQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDENLRIVVADLFSAGMVTTSTTLAWGLLLMILHPDVQRRVQQEIDDVIGQVRRPEMGDQAHMPYTTAVIHEVQRFGDIVPLGVTHMTSRDIEVQGFRIPKGTTLITNLSSVLKDEAVWEKPFRFHPEHFLDAQGHFVKPEAFLPFSAGRRACLGEPLARMELFLFFTSLLQHFSFSVPTGQPRPSHHGVFAFLVSPSPYELCAVPR
SEQ ID NO.14: nucleic acid fragment
GCTTTCCTGCCCCAGCTGGA
SEQ ID NO.15: nucleic acid fragment
CAAAAGGCTTTCCTGCCCCAGCTGGATGAGC
SEQ ID NO.16: nucleic acid fragment
CTTCCAAAAGGCTTTCCTGCCCCAGCTGGATGAGCTGCTA
SEQ ID NO.17: nucleic acid fragment
CTACGCTTCCAAAAGGCTTTCCTGCCCCAGCTGGATGAGCTGCTAACTGA
SEQ ID NO.18: nucleic acid fragment
CGCTTCCAAAAGGCTTTCCTGCCCCAG
SEQ ID NO.19: oligonucleotide fragment
CATCCAGCTGGGGCAG
SEQ ID NO.20: oligonucleotide fragment
CAGCTCATCCAGCTGGGG
SEQ ID NO.21: oligonucleotide fragment
GTTAGCAGCTCATCCAGCTGGGGC
SEQ ID NO.22: oligonucleotide fragment
GCTGGGGCAGGAAAG
SEQ ID NO.23: oligonucleotide fragment
CCAGCTGGGGCAGGAAAGCC 。
Claims (7)
1. for detecting and/or analyze the nucleic acid fragment of CYP2D6 gene single base mutation, described nucleic acid fragment comprises the mutational site of the 2519th corresponding to SEQ ID NO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1 or its reverse complementary sequence, wherein the Nucleotide of the 2519th is C; Or described nucleic acid fragment comprises the mutational site of the 745th corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2 or its reverse complementary sequence, and wherein the Nucleotide of the 745th is C.
2. nucleic acid fragment according to claim 1, is characterized in that, the length of described nucleic acid fragment is 10-100,101-200,201-500 or 501-1000 Nucleotide.
3. nucleic acid fragment according to claim 1, is characterized in that, the length of described nucleic acid fragment is 10-20,21-30,31-40,41-50,51-60,61-100 or 101-300 Nucleotide.
4. nucleic acid fragment according to claim 1, is characterized in that, described nucleic acid fragment is SEQID NO.1,2 or the sequence shown in 14-18.
5., for detecting and/or the test kit of analysis list base mutation, comprising the nucleic acid fragment according to any one of claim 1-4, or comprising the sequence fragment shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.11.
6. the nucleic acid fragment according to any one of claim 1-4 detects the application in the medicine of CYP2D6 transgenation in preparation; Or the application detected in the check mark thing of CYP2D6 transgenation is used as in preparation.
7.CYP2D6 albumen, described protein sequence is the sequence shown in SEQ ID NO.3.
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| Title |
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| Discovery of Novel Functional Variants and Extensive Evaluation of CYP2D6 Genetic Polymorphisms in Koreans;LEE ET AL;《DRUG METABOLISM AND DISPOSITION》;20090413;第37卷(第7期);1464－1470 * |
| Resequencing, haplotype construction and identification of novel variants of CYP2D6 in Mexican Mestizos;Alejandra V Contreras，et al;《Pharmacogenomics》;20110311;第12卷(第5期);745–756 * |
| Zhiyu Zeng， et al.CYP2D6 polymorphisms influence tamoxifen treatment outcomes in breast cancer patients: a meta-analysis.《Cancer Chemother Pharmacol》.2013,第72卷287–303. * |
| 朱志慧.中国少数民族基因组DNA样本库的建立和CYP2D6在维吾尔族人群的多态性分布.《中国优秀硕士学位论文全文数据库，医药卫生科技辑》.2012,(第3期),全文. * |
| 满序聪.中国少数民族基因组DNA样本库的建立和CYP2D6在维吾尔族人群中的多态性分布规律.《中国优秀硕士学位论文全文数据库(电子期刊) 医药卫生科技辑》.2012,全文. * |
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