单管PCR-Pyrosequencing快速检测华法林代谢酶基因多态性方法的建立

文章旨在建立一种单管、快速及高通量的华法林药物代谢酶相关基因多态性的检测方法。通过抽取人外周血DNA,应用带有生物素标记的扩增引物,经PCR扩增并制备焦磷酸测序单链模板,于PyroMark ID焦磷酸测序仪上进行焦磷酸测序,以Sanger测序法测序结果为对照,观察分析的准确性。结果显示,华法林药物代谢酶的3个相关基因多态性(CYP2C9*2、CYP2C9*3、VKORC1(-1693))于单管中可被同时检测,一次可获得96份DNA的华法林药物代谢相关多态性位点检测结果。经与Sanger测序方法比较,符合率为100%。结果表明本方法可准确、高通量、快速检测华法林药物代谢酶相关基因多态性,与单管检测一个位点的焦磷酸测序方法相比,能有效降低检测成本,节省检测时间。该方法在个性化医疗上有较大的推广应用价值,也可以将该平台运用于其他疾病相关基因多态性检测。

Development of a single-tube PCR-pyrosequencing method for simultaneous and rapid detection of the genetic polymorphism of warfarin metabolizing enzymes

The purpose of this article is to develop a new high throughput method for detecting genetic polymorphism of warfarin metabolism-related genes rapidly in a single tube.Genomic DNA from human peripheral blood was extracted,and amplified with biotinylated primer to obtain single-stranded templates for pyrosequencing.Then,the single-stranded templates were subjected to Pyrosequencing analysis using PyroMark ID instrument.Simultaneously,Sanger sequencing was also applied to sequence the products as a control to...