【摘要】 目的建立人端粒保护蛋白1(protection of telomeres 1,POT1)rs1034794位点多态性的检测方法。方法应用创造酶切位点法(created restriction site PCR,CRS-PCR)根据单碱基突变位点的碱基替代情况设计PCR引物。其中1条引物根据突变位点邻近序列设计,引入错配碱基,使得引物3’端和单碱基突变的一种突变型在PCR扩增后形成1个酶切位点,PCR产物用PCR-限制性片段长度多态性(PCR restriction fragment length polymorphism,PCRRFLP)法进行分析。结果设计1对特异引物,其中正向引物3’末端与多态相邻,倒数第三位碱基为错配碱基A可在PCR扩增后与多态T等位基因及相邻片段形成AGCT结构,使用限制性内切酶Alu I酶切效果较好。结论应用CRS-PCR方法创造的酶切位点可以有效而简捷地检测POT1(rs1034794)基因多态性。更多还原

【Abstract】 Objective To establish an appropriate method for identifying single nucleotide polymorphism( SNP) rs1034794 of telomeres protection protein 1( POT1) gene. Methods Created restriction site-PCR( CRS-PCR) was used to design the primers.One of the primers was designed on the basis of neighborhood sequence of the mutational site,the mismatched base was inserted for the purpose of producing a new restriction enzyme cutting site,then PCR products were analyzed by the method of PCR-RFLP( PCR-restriction fragment length polymorphism). Results Specific primers were designed,and the 3’end of the sense primer was adjacent to the polymorphic site,the third base from bottom was the mismatched base A,which can form AGCT structure with allele gene T and neighborhood fragments after PCR amplification,and this structure can be well cut by restriction enzyme Alu I. Conclusion CRSPCR-RFLP method can be used to detect the SNP of POT1 gene,effectively and simply.更多还原