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目的:为了检测信号转导和转录活化蛋白2(signal transducer and activator of transcription factor 2,STAT2)基因的rs12422499和rs2066807两个单核苷酸多态性(single nucleotide polymorphism,SNP),建立一种简单、可靠的多聚酶链式反应-引物介导的限制性分析法(polymerase chain reaction-primer introduced restriction analysis,PCR-PIRA)。方法:微量盐析法提取外周静脉全血DNA,设计引物扩增STAT2基因的rs2066807及rs12422499位点所在的基因片段,采用限制性内切酶PsyI及BseDI分别酶切相应PCR产物,凝胶电泳方法观察酶切结果。同时将PCR产物进行DNA测序。结果:PCR扩增rs2066807基因片段,长度为469bp,经PsyI酶切后电泳,根据酶切产物片段大小判断rs2066807的C/C(469 bp)、G/G(262 bp、207 bp)、C/G(469 bp、262 bp及207bp)三种基因型;PCR扩增rs12422499基因片段,长度为189 bp,经BseDI酶切后电泳,根据酶切产物片段大小判断rs12422499的C/C(189 bp)、G/G(134 bp、55 bp)、C/G(189 bp、134 bp及55 bp)三种基因型。将PCR产物直接进行DNA测序,SNP测序结果与PCR-PIRA方法获得的结果一致。结论:成功建立了一种检测STAT2基因SNPs的PCR-PIRA法,该方法只需简单的PCR扩增和酶切消化,因此操作简单、方便;通过与直接测序结果比较,显示该方法可信。PCR-RIPA方法不仅为检测STAT2基因多态性提供了实验手段,也为检测其他基因点突变提供了实验方法。
OBJECTIVE: To detect single nucleotide polymorphisms (SNP) of rs12422499 and rs2066807 in signal transducers and activator of transcription factor 2 (STAT2) gene, a simple , Reliable polymerase chain reaction-primer introduced restriction restriction analysis (PCR-PIRA). Methods: Peripheral venous whole blood DNA was extracted by micro salting-out method. Primers were designed to amplify the gene fragment of rs2066807 and rs12422499 of STAT2 gene. Restriction endonucleases PsyI and BseDI were respectively used to digest the corresponding PCR products and gel electrophoresis Observe the results of digestion. The PCR product was simultaneously DNA sequenced. Results: The rs2066807 gene fragment was 469 bp in length and was digested with PsyI. The fragment of rs2066807 was identified as C / C (469 bp), G / G (262 bp, 207 bp) and C / G (469 bp, 262 bp and 207 bp). The rs12422499 gene fragment was 189 bp in length and was sequenced by BseDI electrophoresis. The C / C (189 bp) value of rs12422499 was determined according to the size of the digested fragment. , G / G (134 bp, 55 bp), C / G (189 bp, 134 bp and 55 bp). PCR products were directly DNA sequencing, SNP sequencing results and PCR-PIRA method results obtained. CONCLUSION: A PCR-PIRA method for detecting SNPs of STAT2 gene has been successfully established. This method requires only simple PCR amplification and restriction enzyme digestion, so it is simple and convenient to operate. Compared with the direct sequencing results, it shows that the method is credible. The PCR-RIPA method not only provides an experimental method for detecting STAT2 gene polymorphism, but also provides experimental methods for detecting point mutations of other genes.