乙醛脱氢酶2基因G1510A多态位点等位基因的克隆及应用

Cloning and application of alleles of acetaldehyde dehydrogenase 2 gene G1510A polymorphic locus

目的克隆乙醛脱氢酶2(ALDH2)基因G1510A多态位点(rs671位点)的野生型和突变型等位基因,为该位点的基因分型提供参照品。方法分别以经测序证实的rs671位点野生型纯合子和突变型纯合子样品为模板,PCR扩增跨越该位点的DNA片断,采用胶回收试剂盒纯化PCR产物,T-A克隆构建重组质粒,PCR和测序验证重组质粒是否构建成功,PCR-RFLP法检验其应用效果。结果 PCR和测序均证实目的基因被成功克隆,将重组质粒用于PCR-RFLP法基因分型之中,能正确区分样品的基因型。结论成功构建了rs671位点的野生型和突变型等位基因重组质粒,可用作该位点基因分型的参照品。

Objective To clone the wild and mutant alleles of acetaldehyde dehydrogenase 2 gene G1510A polymorphic locus for providing control samples for genotyping this locus.Methods The fragments spanning the G1510A locus were amplified by PCR with the template DNA from wild and mutant homozygotes.PCR products were purified with the gel extraction purification kit and cloned into pMD18-T vector.PCR and sequencing were taken to verify whether the recombinant plasmids were constructed successfully.The application effect of recombinant plasmid was examined by PCR-RFLP.Results PCR and sequencing showed that the target gene was successfully cloned.Genotypes were distinguished correctly by PCR-RFLP using the recombinant plasmid as reference.Conclusion The recombinant plasmid containing wild and mutant alleles of rs671 locus has been successfully constructed and can be used as a reference for genotyping.