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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、特异的CYP2A6基因多态性分析法
Objective: To establish a one-step PCR RFLP assay for CYP2A6 gene polymorphism. Methods: peripheral blood 10 0 μl, extracted with Rose method genomic DNA, using specific primers CYP2A6F0 3: 5 ’CTGATCGACTAGGCGTGGTA 3’, CYP2A6R0 6: 5 ’CGTCCTGGGTGTTTTCCTTC 3’ PCR amplification step carried out with restriction endonuclease Dde I, Xcm I PCR products were digested, 3% agarose gel electrophoresis. Results: The size of amplified PCR product was 2 14bp. Some of the blood samples did not get the PCR amplification product, which was judged as CYP2A6 deletion genotype (CYP2A6del / CYP2A6del). DdeⅠ After digestion, gel electrophoresis obtained partially digested (CYP2A6wt / CYP2A6v2 genotype), is not digested (CYP2A6wt / CYP2A6wt genotype) of two types of DNA fragments; XcmⅠ After digestion, gel electrophoresis obtained partzyme Cut (CYP2A6wt / CYP2A6v1 genotype), not digested (CYP2A6wt / CYP2A6wt genotype) two types of DNA fragments. Among them, some blood samples were digested by both Dde I and Xcm I (CYP2A6v2 / CYP2A6v1 genotype). Application of this method testing found that Chinese patients with gastric cancer CYP2A6 allele polymorphism exists. Double-blind repeated testing, the above results are consistent. Conclusion: The simple, stable and specific CYP2A6 gene polymorphism can be established by one-step PCR RFLP