CN105506092A - Primer of CYP2D6_C100T gene polymorphism and detection method thereof - Google Patents
Primer of CYP2D6_C100T gene polymorphism and detection method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, in particular relates to a primer of CYP2D6_C100T gene polymorphism and a detection method thereof. The primer of the CYP2D6_C100T gene polymorphism comprises a nested PCR (polymerase chain reaction) amplification primer and an SNaPshot PCR primer; the nested PCR amplification primer comprises a nested A-round PCR amplification primer and a nested B-round PCR amplification primer. The primer of the CYP2D6_C100T gene polymorphism has the advantages of good specificity, no cross reaction and high accuracy. The detection of the CYP2D6_C100T gene polymorphism is realized, and the primer has important significance for realizing the gene oriented individualized treatment, continuously improving the treatment effect and reducing the adverse reaction.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of primer and detection method thereof of CYP2D6_C100T gene pleiomorphism.
Background technology
CYP2D6 is first and is identified by the P450 enzyme of Dominant gene, and CYP2D6 is positioned on No. 22 karyomit(e), and it contains 9 exons and 8 introns, and overall length is about 7kb, is a complete functioning gene.Studies have found that, CYP2D6 upstream region of gene has the pseudogene of 2 very high homology, be called CYP2D7P gene and CYP2D8P gene, CYP2D7P gene and CYP2D6 gene have the nucleic acid sequence homology of 97%, and with TATA box, but owing to inserting a thymus pyrimidine (T) on the 226th site of exons 1, thus change single open reading frame, make premature transcription termination; CYP2D8P gene is a sudden change pseudogene containing multiple breaking point.CYP2D7P and CYP2D8P gene is not all expressed in human footprint, only has CYP2D6 to express in liver, intestines, kidney and human brain.
Modern study finds, in liver, the content of CYP2D6 only accounts for 2% of P450 liver protein total amount, but it but participates in the clinical application of metabolism about 25%, comprises thymoleptic, anti-arrhythmic, antipsychotic drug and anodyne etc.Find the allelic variation of CYP2D6 more than 100 kinds at present; the mainly loss of single nucleotide variations, large fragment gene and polygene copy; these variations make CYP2D6 gene present polymorphism; and determine the diversity of its metabolic phenotype, make the activities present of enzyme be disappearance, reduce, the several types such as normal or increase.
The people such as Yang Xi have delivered the paper that one section of exercise question is " CYP2D6 gene pleiomorphism and tamoxifen are treated ", this paper shows that the CYP2D6 of different genotype is different to the metabolism of tamoxifen, active metabolite (4-hydroxytamoxifen and the Endoxifen) concentration produced is different, wherein in PMs (causing CYP2D6 activity disappearance containing two amorphss) blood plasma, active metabolite concentration is minimum, and UMs (containing two or more normal function allelotrope, namely polygene copy causes CYP2D6 increased activity) plasma activities Metabolites Concentration is the highest.Therefore, the polymorphism research of CYP2D6 has great importance to clinical, becomes the focus of research at present.
Summary of the invention
For solving problems of the prior art, the invention provides a kind of primer and detection method thereof of CYP2D6_C100T gene pleiomorphism, this primer detects CYP2D6 gene C 100T (rs1065852) site, have that specificity is good, highly sensitive, accuracy advantages of higher, for CYP2D6_C100T gene pleiomorphism provides a kind of simple and effective detection method.
The invention provides a kind of primer of CYP2D6_C100T gene pleiomorphism, comprise nested PCR amplification primer and SNaPshotPCR primer; Described nested PCR amplification primer comprises nido A and takes turns pcr amplification primer and nido B wheel pcr amplification primer; Described nido A takes turns amplimer: for upstream primer 5 '-CCAGAAGGCTTTGCAGGCTTCA-3 ' (SEQIDNO.1) and the downstream primer 5 '-CTGAGCCCTGGGAGGTAGGTAG-3 ' (SEQIDNO.2) of CYP2D6; Described nido B takes turns pcr amplification primer: for upstream primer 5 '-CAGAGGAGCCCATTTGGTAG-3 ' (SEQIDNO.3) and the downstream primer 5 '-CCTGGTCGAAGCAGTATGGT-3 ' (SEQIDNO.4) of CYP2D6_C100T; Described SNaPshotPCR primer is: for CYP2D6_C100TSNaPshotPCR primer 5 '-ACGCTGGGCTGCACGCTAC-3 ' (SEQIDNO.5).
In addition, present invention also offers a kind of detection method of CYP2D6_C100T gene pleiomorphism, comprise the steps:
S1 extracts DNA sample;
S2 adopts nido A according to claim 1 wheel pcr amplification primer to carry out pcr amplification, takes turns pcr amplification product carry out purifying to nido A;
S3 takes turns pcr amplification product with nido A and carries out multiplexed PCR amplification for template adopts the nido B described in claim to take turns pcr amplification primer;
S4 adopts SNaPshotPCR primer according to claim 1 to carry out SNaPshotPCR amplification, carries out purifying to amplified production;
S5 capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
Further, the DNA sample in described step S1 is the DNA sample of EDTA anticoagulation cirumferential blood.
Further, the nido A in described step S2 takes turns pcr amplification reaction condition and comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:72 DEG C 3min; Stage 4: turn back to stage 2,24 circulation; Stage 5:72 DEG C 5min; Stage 6:25 DEG C of insulation.
Further, the nido B in described step S3 takes turns pcr amplification reaction condition and comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.
Further, the SNaPshotPCR amplification reaction condition in described step S4 comprises: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
Further, GENEMAPPERIDV4.1 software is adopted to analyze detected result in described step S5.
In addition, the primer of CYP2D6_C100T gene pleiomorphism provided by the invention detects the purposes in CYP2D6_C100T gene pleiomorphism reagent in preparation.
Compared with prior art, technical scheme provided by the invention has following advantage: the primer that the invention provides a kind of CYP2D6_C100T gene pleiomorphism, the specificity of this primer is good, cross reaction can not be there is, accuracy is good, achieve the detection of CYP2D6_C100T gene pleiomorphism, to realizing gene targeting individualized treatment, improve constantly result for the treatment of, reduction untoward reaction has very important significance.
Accompanying drawing explanation
Fig. 1 is the amplified fragments of CYP2D6_C100T provided by the invention;
Fig. 2 is the part sequencer map of CYP2D6_C100T primer extension product;
Fig. 3 is the CYP2D6_C100T genetic polymorphism detection result figure of sample.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
Embodiment one, primer
Primer provided by the invention is as shown in table 1, comprises nested PCR amplification primer and SNaPshotPCR primer; Described nested PCR amplification primer comprises nido A and takes turns pcr amplification primer and nido B wheel pcr amplification primer, and described nido B takes turns pcr amplification primer and SNaPshotPCR primer is corresponding.All primer sequences provided by the invention all by the comparison of UCSC database, without known SNP site.
Table 1 primer provided by the invention
The specificity of embodiment two, primer
Primer provided by the invention is carried out Blasting in UCSC, and result is as follows:
CYP2D6_C100T gene specific primer carries out Blast in UCSC, without other homologous gene.CYP2D6_C100T amplified fragments is positioned at chr22:42130611-42130821, and length is 211bp, and extension increasing sequence is as Fig. 1.
In use table 1, pcr amplification primer increases and Sanger order-checking to detection sample respectively, and sequencing result shows, and each primer amplification fragment is coincide with CYP2D6_C100T gene reference sequence, and result as shown in Figure 2.Use SNaPshotPCR primer in table 1, SNaPshot method detects, and result as shown in Figure 3.As can be seen from Figure 3, the base that the relative position at each product peak and sequencing reaction mix conforms to expection, and without other Interference Peaks.
The detection of embodiment three, CYP2D6_C100T gene pleiomorphism
1) extract the DNA sample of EDTA anticoagulation cirumferential blood, extracting method with reference to TIANampBloodDNAKit(purchased from Tiagen, article No. DP318) specification sheets, DNA sample is diluted to 100ng/ μ L, for subsequent use.
2) prepare primer mixture: preparation nido A takes turns PCR primer mixture, CYP2D6-Fwd:CYP2D6-Rev:H2O=1:1:3, and primer final concentration is 1pmol/ μ L, concussion mixing is of short duration centrifugal rear for subsequent use; Preparation B takes turns PCR primer mixture, and primer final concentration is 0.5pmol/ μ L; Of short duration centrifugal rear for subsequent use; Pcr amplification adopts Q5
?warm start surpasses fidelity 2XMasterMix(purchased from NEB company, article No. M0494L), reaction system is as shown in table 2.A takes turns pcr amplification reaction condition and comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:72 DEG C 3min; Stage 4: turn back to stage 2,24 circulation; Stage 5:72 DEG C 5min; Stage 6:25 DEG C of insulation.Nido B takes turns PCR and takes turns PCR primer for template with A, and nido B takes turns pcr amplification reaction condition and comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.After pcr amplification terminates, get 2.0 μ LExoSAP-IT(purchased from Affymetrix, article No. 78205) and 3.0 μ LddH
2o, mixing, adds pcr amplification product 2.0 μ L, mixes micro-centrifugal; Endonuclease reaction is carried out, program: 37 DEG C, 15min in PCR instrument; 80 DEG C, 15min; 4 DEG C, insulation.
Table 2, PCR reagent preparation form
3) configure SNaPshotPCR primer mixture, primer final concentration is 0.8pmol/ μ L, adopts SNaPshot
?multiplexKit(purchased from ABI company, article No. 4323151) increase, reaction system is as shown in table 3.SNaPshotPCR reaction conditions comprises: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
Table 3, SNaPshot preparation of reagents form
In SNaPshotPCR product, add 1.0 μ LSAP enzymes, react according to following program: 37 DEG C, 15min; 80 DEG C, 15min; 4 DEG C, insulation.After completion of the reaction, capillary electrophoresis technique detects, and adopt GENEMAPPERIDV4.1 software to analyze to detected result, determine SNP site and genotype thereof, result as shown in Figure 3.
The specificity of the method for embodiment four, detection CYP2D6_C100T gene pleiomorphism
The specificity of the inventive method is defined as negative match-rate.The present invention is optimized SNaPshot sequencing to 22 routine samples and detects, and detected result adopts Sanger sequencing to verify.The result that the negative findings that SNaPshot sequencing detects shows with Sanger method conforms to (see table 4).Detection specificity of the present invention is 100%.
Table 4, CYP2D6_C100T detection specificity testing data
The sensitivity of the method for embodiment five, detection CYP2D6_C100T gene pleiomorphism
The sensitivity definition of the inventive method is positive coincidence rate.The present invention is optimized SNaPshot sequencing to 22 routine samples and detects, and detected result adopts Sanger sequencing to verify.The result that the positive findings that SNaPshot sequencing detects shows with Sanger method conforms to (see table 5).Detection sensitivity of the present invention is 100%.
Table 5, CYP2D6_C100T detection sensitivity
The accuracy of the method for embodiment six, detection CYP2D6_C100T gene pleiomorphism
Accuracy of the present invention is defined as the consistence of different methods detected result.The present invention carries out the detection of SNaPshot sequencing to 22 routine samples altogether, and detected result all adopts Sanger sequencing to verify.Different methods detected result is consistent, as shown in table 6.Accuracy of the present invention is 100%.
Table 6, CYP2D6_C100T gene SNP s site accuracy in detection
The precision of the method for embodiment seven, detection CYP2D6_C100T gene pleiomorphism
Precision of the present invention is defined as carries out to sample the ability that duplicate detection obtains same result.Invention has been between personnel, simultaneous test (see table 7) between the different hole of different time, different instrument, same sample, all results all show unanimously, and precision of the present invention is 100%.
Table 7, betweenrun precision testing data
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
SEQUENCELISTING
Science and Technology Co., Ltd. is detected in gold territory, <110> Guangzhou
The primer of a <120> CYP2D6_C100T gene pleiomorphism and detection method thereof
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ccagaaggctttgcaggcttca22
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ctgagccctgggaggtaggtag22
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cagaggagcccatttggtag20
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cctggtcgaagcagtatggt20
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acgctgggctgcacgctac19
Claims (8)
1. a primer for CYP2D6_C100T gene pleiomorphism, is characterized in that: comprise nested PCR amplification primer and SNaPshotPCR primer; Described nested PCR amplification primer comprises nido A and takes turns pcr amplification primer and nido B wheel pcr amplification primer; Described nido A takes turns amplimer: for upstream primer 5 '-CCAGAAGGCTTTGCAGGCTTCA-3 ' and the downstream primer 5 '-CTGAGCCCTGGGAGGTAGGTAG-3 ' of CYP2D6; Described nido B takes turns pcr amplification primer: for upstream primer 5 '-CAGAGGAGCCCATTTGGTAG-3 ' and the downstream primer 5 '-CCTGGTCGAAGCAGTATGGT-3 ' of CYP2D6_C100T; Described SNaPshotPCR primer is: for CYP2D6_C100T_SNaPshotPCR primer 5 '-ACGCTGGGCTGCACGCTAC-3 '.
2. a detection method for CYP2D6_C100T gene pleiomorphism, is characterized in that: comprise the steps:
S1 extracts DNA sample;
S2 adopts nido A according to claim 1 wheel pcr amplification primer to carry out pcr amplification, takes turns pcr amplification product carry out purifying to A;
S3 takes turns pcr amplification product with nido A, and for template adopts, nido B wheel pcr amplification primer according to claim 1 carries out multiplexed PCR amplification;
S4 adopts SNaPshotPCR primer according to claim 1 to carry out SNaPshotPCR amplification, carries out purifying to amplified production;
S5 capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
3. the detection method of CYP2D6_C100T gene pleiomorphism according to claim 2, is characterized in that: the DNA sample in described step S1 is the DNA sample of EDTA anticoagulation cirumferential blood.
4. the detection method of CYP2D6_C100T gene pleiomorphism according to claim 2, is characterized in that: the nido A in described step S2 takes turns pcr amplification reaction condition and comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:72 DEG C 3min; Stage 4: turn back to stage 2,24 circulation; Stage 5:72 DEG C 5min; Stage 6:25 DEG C of insulation.
5. the detection method of CYP2D6_C100T gene pleiomorphism according to claim 2, is characterized in that: the nido B in described step S3 takes turns pcr amplification reaction condition and comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.
6. the detection method of CYP2D6_C100T gene pleiomorphism according to claim 2, is characterized in that: the SNaPshotPCR amplification reaction condition in described step S4 comprises: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
7. the detection method of CYP2D6_C100T gene pleiomorphism according to claim 2, is characterized in that: adopt GENEMAPPERIDV4.1 software to analyze detected result in described step S5.
8. the primer of CYP2D6_C100T gene pleiomorphism according to claim 1 detects the purposes in CYP2D6_C100T gene pleiomorphism reagent in preparation.
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2015
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| CN1616675A (en) * | 2003-09-22 | 2005-05-18 | 周宏灏 | Individual administration gene type diagnostic chip and its producing method and using method |
| CN101054601A (en) * | 2006-04-13 | 2007-10-17 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Oligonucleotide for detecting cytochrome P450 enzyme series mutation site and gene chip |
| CN101824467A (en) * | 2009-12-29 | 2010-09-08 | 广州益善生物技术有限公司 | CYP2D6 gene mutation detection liquid-phase chip and detection method |
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| Title |
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| A.TOUATI等: "Molecular Epidemiology of Mycoplasma pneumonia: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| J.萨姆布鲁克等: "《分子克隆实验指南（上册）》", 31 August 2002, 科学出版社 * |
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| NELSON D.R等: "Homo sapiens cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) on chromosome 22", 《GENBANK DATABASE》 * |
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| 杨汐等: "CYPD2D6基因多态性与他莫昔芬治疗", 《西南军医》 * |
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