应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型  

Duplex genotyping of CYP2C19*2 and CYP2C19*3 by high-resolution melting curve analysis

作　　者：曹春鸽[1] 孙海燕[1] 周芳芳[1] 王诗铭[1] 陈红岩[1] 卢大儒[1] 

机构地区：[1]复旦大学生命科学学院,遗传工程国家重点实验室,上海200433

出　　处：《遗传》2013年第7期923-930,共8页Hereditas（Beijing)

基　　金：国家高技术研究发展计划(863计划)项目(编号:2012AA02A517;2012AA02A518)资助

摘　　要：氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明,携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人,其体内代谢氯吡格雷成为其活性形式的能力降低,导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curveanalysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物,并在两个位点引物的5′端分别加上富含AT和GC的序列,保证两个位点的扩增产物熔解峰无重叠。利用HRM技术,快速、灵敏地对64例随机DNA样本的CYP2C19*2、CYP2C19*3两个多态性位点进行了基因分型,且HRM方法的分型结果与测序验证结果完全一致。因此,利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床,指导氯吡格雷的个体化用药。Clopidogrel is a widely used anti-platelet agent for the prevention of arterial thrombosis. It has been sug- gested that clopidogrel may be less effective in inhibiting platelet aggregation among patients who are carriers of CYP2C19＊2 and CYP2C19＊3, two loss-of-function CYP2C19 alleles, which are associated with reduced conversion of clopidogrel to its active metabolite. The objective of this research was to develop a simple and accurate method for geno- typing of CYP2C19＊2 and CYP2C19＊3 simultaneously in one closed-tube using high-resolution melting curve （HRM） analysis. Two amplicons bracketing CYP2C19＊2 and CYP2C19＊3 gene variants were designed, and AT- or GC-rich 5＇ tailswere added to selected primers to ensure two different amplicons with non-overlapping melting curves. Sixty-four random DNA samples were all fast and sensitively genotyped by HRM analysis. This method was validated by DNA sequencing- technique, and genotypes obtained using the HRM approach perfectly matched the genotypes obtained by DNA sequencing technique. Therefore, this HRM-based assay allows simple and accurate duplex genotyping of CYP2C19＊2 and CYP2C19＊3 simultaneously in one closed-tube. This method is expected to be applied in clinical laboratory to guide indi- vidual dosage design of clopidogrel.

关 键 词：高分辨率熔解曲线分析 CYP2C19＊2 CYP2C19＊3 氯吡格雷 SNP分型 

分 类 号：R972[医药卫生—药品]