摘要
目的 :建立CYP2A6基因多态性的一步PCR RFLP分析法。方法 :取外周静脉血 10 0 μl,用Rose法提取基因组DNA ,用特异性引物CYP2A6F0 3:5’ CTGATCGACTAGGCGTGGTA 3’ ,CYP2A6R0 6 :5’ CGTCCTGGGTGTTTTCCTTC 3’进行一步PCR扩增 ,用限制性内切酶DdeⅠ ,XcmⅠ对PCR产物进行酶切 ,3%琼脂糖凝胶电泳。结果 :扩增的PCR产物大小为 2 14bp ,部分血样标本未获得PCR扩增产物 ,判为CYP2A6缺失基因型(CYP2A6del/CYP2A6del)。DdeⅠ酶切后 ,凝胶电泳可得到部分酶切 (CYP2A6wt/CYP2A6v2基因型 ) ,未被酶切(CYP2A6wt/CYP2A6wt基因型 )两种类型DNA片段 ;XcmⅠ酶切后 ,凝胶电泳可得到部分酶切 (CYP2A6wt/CYP2A6v1基因型 ) ,未被酶切 (CYP2A6wt/CYP2A6wt基因型 )两种类型DNA片段。其中 ,部分血样标本同时被DdeⅠ和XcmⅠ部分酶切 (CYP2A6v2 /CYP2A6v1基因型 )。应用此方法检测 ,发现中国人群胃癌患者存在CYP2A6等位基因多态性。经双盲重复检测 ,上述结果一致。结论 :采用一步PCR RFLP技术可以建立简单、稳定。
Objective: To set up one-step method for analysis of CYP2A6 gene polymorphism. Methods:Genomic DNA was extracted from peripheral vein blood by ROSE method:PCR amplification was done with specific primers: CYP2A6F03:5’-CTG ATC GAC TAG GCG TGG TA-3’,CYP2A6R06:5’-CGT CCT GGG TGT TTT CCT TC-3’. PCR products were digested with restricted enzymes DdeI, XcmI, respectively and run electrophoresis on 3% agarose gel. Results: PCR product was 214 bp, some samples could’t be amplicated by specific primers.They were deletional genotype of CYP2A6 gene(CYP2A6del/CYP2A6del).Two kinds of DNA fragment were seen when PCR products digested with DdeI and were run on 3% agarose gel, part digestion(CYP2A6wt/CYP2A6v2 genotype) and no digestion(CYP2A6wt/CYP2A6wt genotype);Two kinds of DNA fragment were seen when PCR products digested with XcmI and run on 3% agarose gel,part digestion(CYP2A6wt/CYP2A6v1 genotype) and no digestion(CYP2A6wt/CYP2A6wt genotype). In this study,some samples’ PCR products were digested by both DdeI and XcmI(CYP2A6v2/CYP2A6v1 genotype). There was CYP2A6 gene polymorphism in Chinese patients with gastric cancer. All results were confirmed repeatedly by double-blind analysis. Conclusion: One-step PCR-RFLP method is useful for analysis of CYP2A6 gene polymorphism.
出处
《江西医学院学报》
CAS
2002年第5期23-25,27,共4页
Acta Academiae Medicinae Jiangxi
基金
教育部国家留学基金委 (CSC)资助 (学号 :98836 0 0 7
