高分辨率熔解曲线法在GLP1R基因多态性位点rs3765467检测中的应用
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篇名: 高分辨率熔解曲线法在GLP1R基因多态性位点rs3765467检测中的应用
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摘要: 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39 065 819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39 065 817和39 065 819两个多态性位点。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。
ABSTRACT: 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: 39 065 819), 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 resolution melting (HRM) method was adopted to analyze amplified product. Sequencing verification by double stranded chain termination 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 39 065 817 and 39 065 819 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 mutation, economical and simple method should be selected after comprehensive analysis of sequence.
期刊: 2017年第28卷第17期
作者: 张远,何霞,钟磊,串俊兰,龙恩武
AUTHORS: ZHANG Yuan,HE Xia,ZHONG Lei,CHUAN Junlan,LONG Enwu
关键字: GLP1R基因;高分辨率熔解曲线;单核苷酸多态性;基因检测
KEYWORDS: GLP1R gene; High resolution melting; Single nucleotide polymorphism; Gene detection
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