检测STAT2基因多态性的PCR-PIRA方法的建立  

Establishment of PCR-PIRA for Detection of STAT2 Gene Polymorphism

作　　者：袁媛[1,2] 黄玉梅[1] 钟待鸣[2] 朱敏[3] 姚芳玲[1] 黎明[1] 

机构地区：[1]中南大学基础医学院免疫学教研室,湖南长沙410078 [2]长沙血液中心,湖南长沙410011 [3]中南大学生物科学与技术学院分子生物学研究中心,湖南长沙410078

出　　处：《现代生物医学进展》2013年第14期2763-2767,共5页Progress in Modern Biomedicine

基　　金：湖南省卫生厅科技基金项目(132012-137)

摘　　要：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： In order to detect two Signal Nucleotide Polymorphisms（SNPs）,rs12422499 and rs2066807 gene of Signal Transducer and Activator of Transcription Factor 2（STAT2）,a simple and reliable method,named polymerase chain reaction-primer introduced restriction analysis（PCR-PIRA） was established.Methods： DNA from peripheral blood was extracted using the trace salting-out method.Design PCR primers to amplify the gene fragment containing STAT2 SNP,rs2066807 or rs12422499,respectively.Digest the corresponding PCR product by PsyI or BseDII.Separate the digested products by electrophoresis.And then,the PCR products were sequenced.Results： The length of PCR product of the gene fragment containing rs2066807 was 469 bp.This product was digested by PsyI following by electrophoresis.According to the electrophretic maps,three genotypes of rs2066807 which were C/C（469 bp）,G/G（262 bp and 207 bp） and C/G（469 bp,262 bp and 207 bp） were decided.The length of PCR product of the gene fragment containing rs12422499 was 189 bp.This product was digested by BseDI following by electrophoresis.According to the electrophretic maps,three genotypes of rs12422499 which were C/C（189 bp）,G/G（134 bp and 55 bp）,and C/G（189 bp,134 bp and 55 bp） were decided,The PCR products were sequenced.The sequencing results of the two SNPs were in good agreement with that detected by PCR-PIRA.Conclusion： The PCR-PIRA method for detection of SNPs of STAT2 was established.The procedure of PCR-PIRA is very simple and convenient,only including a PCR amplification followed by digestion by an enzyme.The sequencing results of the two SNPs were consistent with that detected by PCR-PIRA,demonstrating that PCR-RIPA is reliable.The strategy of PCR-PIRA does not only offer an experimental method for detection of polymorphism of STAT2,but also a mean of gene point mutation.

关 键 词：多聚酶链式反应-引物介导的限制性分析法 单核苷酸多态性 信号转导和转录活化蛋白2 

分 类 号：R-33[医药卫生] R446.6