重庆医学Issue(10)：1370-1373,1377,5.DOI:10.3969/j.issn.1671-8348.2015.10.026
基于连接酶-琼脂糖凝胶电泳的单核苷酸多态性快速检测方法
A rapid detection method for single nucleotide polymorphisms based on ligase-agarose gel electrophoresis
摘要
Abstract
Objective To establish a simple,rapid and sensitive nucleotide polymorphisms genotyping method in order to conduct the routine clinical detections under the simple laboratory condition by this method.Methods Based on the ligase-agarose gel electrophoresis,the oligonucleotide detection probes of mutational sites was designed.The detection underwent the detection probe connecting,purification and universal amplification,finally the mutation genotypes of detection sites were judged by the ap-peared bands in the agarose gel electrophoresis(AGE).With the 3 SNP sites EGFR,c.2573T>G(L858R),EGFR,c.2582T> A (L861Q)and EGFR,c.2155 G>T(G719C)in epidermal growth factor receptor(EGFR)gene as the detection objects,the plasmid template and plasma circulating DNA sample in lung cancer were performed the detection.Results The established method was easy to operate with higher specificity and sensitivity.After 20-30 cycles of PCR amplification,the genotype of detection sites was clearly estimated according to the amplification band.When detecting the mixed alleles in the heterogeneous sample,minimal 2.5%mutation alleles could be detected out.This method and the direct sequencing method could respectively detect 6 cases and 2 cases of heterozygotes mutation in the SNP site of L858R among 62 samples of lung cancer.Conclusion The established detection method for SNP genotyping is suitable to the routine mutation detection on the heterogeneous samples under the simple laboratory condi-tion.关键词
多态性,单核苷酸/突变/基因型/连接酶类/电泳,琼脂凝胶Key words
polymorphism,single nucleotide/mutation/genotype/ligases/electrophoresis,agar gel分类
医药卫生引用本文复制引用
崔海忠,肖娜,张永平,陈大贵,唐一通,赵雪红,邵金辉..基于连接酶-琼脂糖凝胶电泳的单核苷酸多态性快速检测方法[J].重庆医学,2015,(10):1370-1373,1377,5.基金项目
湖北省卫生计生科研基金（WJ2015MB266）；湖北省教育厅资助项目（Q20132604）；襄阳市科技局资助项目［襄科业（2012）43号］；湖北省教育厅高校青年教师深入企业计划项目（2014）。 （）
