目的:建立检测肠促胰岛素样肽-1受体(GLP1R)基因已知突变位点rs3765467(NT_007592.16的第39065819位)的方法,并评价其准确性与实用性.方法:收集我院体检中心2015年10月-2016年2月72例健康体检者的外周静脉血样本,采用柱提法提取其全血DNA,经降落聚合酶链反应扩增后,采用高分辨率熔解曲线(HRM)法对产物进行分析;同时选取其中38例受试样本进行双脱氧链终止法(Sanger测序法)测序验证,比较2种方法的结果.结果:突变扫描结果显示,扩增片段中存在39065817和39065819两个多态性位点.HRM法只检测出了4种基因型[GCG/GCG、GCA/GCG或ACG/GCG、GCA/GCA或ACG/ACG、A(G)CA(G)];而Sanger测序法共检测出6种基因型[GCG/GCG、ACG/GCG、ACG/ACG、A(G)CA(G)、GCA/GCG、GCA/GCA].结论:HRM法可区分GCG/GCG和A(G)CA(G)基因型,但无法区分GCA/GCG与ACG/GCG杂合突变、GCA/GCA与ACG/ACG纯合突变.该方法并不适用于多个邻近位点的单核苷酸多态性检测.在进行单核苷酸突变检测时,应对序列进行综合分析后再选取经济、简便的方法.%OBJECTIVE:To establish the method for the detection of the known glucagon-like peptide 1 receptor (GLP1R) gene mutation site rs3765467(NT_007592.16,position:39065819),and to evaluate its accuracy and practicability. METHODS:Peripheral venous blood samples of 72 healthy subjects were collected in medical examination center of our hospital during Oct. 2015-Feb. 2016. The whole blood DNA was extracted by column extraction method. After amplified by touch down PCR,high reso-lution melting(HRM)method was adopted to analyze amplified product. Sequencing verification by double stranded chain termina-tion method(Sanger sequencing method)was performed for 38 test samples. The results of 2 methods were compared. RESULTS:The results of mutation scanning showed that there were 39065817 and 39065819 polymorphism sites in amplified segments. Four types of mutations were detected by HRM method [GCG/GCG,GCA/GCG or ACG/GCG,GCA/GCA or ACG/ACG,A(G) CA(G)],but 6 types of mutations was detected by Sanger sequencing method [GCG/GCG,ACG/GCG,ACG/ACG,A(G)CA (G),GCA/GCG,GCA/GCA]. CONCLUSIONS:HRM method can identify GCG/GCG and A(G)CA(G)genotype,but can not identify GCA/GCG and ACG/GCG heterozygous mutation,GCA/GCA and ACG/ACG homozygous mutation. The method is not suitable for the detection of single nucleotide polymorphism for multiple neighboring sites. In the detection of single nucleotide mu-tation,economical and simple method should be selected after comprehensive analysis of sequence.
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