SYBR greenⅠ结合熔解曲线分析快速检测GSTM1基因多态性  

Rapid detection of GSTM1 polymorphisms using SYBR green I and melting curve analysis

作　　者：王小飞[1] 王惠民[1] 王跃国[1] 施英娟[1] 张冬雷[2] 

机构地区：[1]南通大学附属医院基因诊断实验室,江苏南通226001 [2]南通大学附属医院酶学研究室,江苏南通226001

出　　处：《临床检验杂志》2006年第2期100-102,共3页Chinese Journal of Clinical Laboratory Science

摘　　要：目的建立一种快速检测人类谷胱甘肽S转移酶(GST)基因多态性的方法并初步应用。方法反应体系中加入新型荧光染料SYBR greenⅠ,应用多重PCR同时扩增GSTM1基因及内参照CYP1A1基因。PCR反应结束后,以0.1℃/s的速度从65℃到95℃检测并记录反应管荧光的变化,根据得到的熔解曲线分析GSTM1基因型。结果GSTM1基因携带型(GSTM1+)熔解曲线出现两个峰带,GSTM1基因空白型(GSTM1-)只出现一个峰带,两峰峰尖所对应的温度值与预期值相同,琼脂糖凝胶电泳显示PCR产物分子量与预期分子量一致。DNA抽提后整个检测在L ightCyc ler扩增仪上1 h内完成。结论SYBR greenⅠ结合熔解曲线分析是一种简单、快速、准确的分析GSTM1基因多态性的方法。Objective To establish a method to detect the glutathione s-transferase（GST） M1 gene polymorphisms rapidly and to apply preliminary for detection. Methods GSTM1 gene and internal control gene CYP1AI were amplified by multi-PCR with appropriate fluorescent dye SYBR green I in the reaction system,The changes of fluorescence values were recorded from 65 ℃ to 95 ℃ by a rate of 0.1℃/s when PCR was just completed. Results There were two peaks in the melting curve of GSTMI ＋ genotype, but only a single peak occurred for the GSTMl-genotype. The temperatures of two peaks corresponded to the expected Tm. agarose gel electrophoresis analysis demonstrated that these peaks correspond to the bands of the predicted molecular size. The detection can be completed within an hour after DNA was extracted. Conclusion Melting curve analysis combined with SYBR green Ⅰ is a easy, rapid, accurate methodfor detection of GSTM1 gene polymorphisms.

关 键 词：SYBR green Ⅰ 熔解曲线分析 基因 多态性 

分 类 号：Q78[生物学—分子生物学]