Pyrosequencing检测CYP2C9^＊3基因多态性方法的建立及其可靠性研究

Development of pyrosequencing method for the simultaneous and rapid detection of CYP2C9^＊3 gene polymorphism and its reliability study

目的:建立基于焦磷酸测序技术(Pyrose-quencing)的高通量的CYP2C9*3突变检测方法。方法:应用带有生物素标记的扩增引物,经PCR扩增及Beads分离,制备该突变位点焦磷酸测序单链模板,并在PyroMark ID焦磷酸测序仪上进行焦磷酸测序,以经典ABI测序法测序结果作为标准对照,观察批量分析的可靠性并批量检测220例人DNA标本。结果:建立了基于焦磷酸测序技术的CYP2C9*3突变位点检测平台,实现了DNA标本的高通量检测。能一次获得96份DNA的CYP2C9*3突变位点检测结果。经重复性检测和标准的ABI测序可靠性检测,CYP2C9*3突变位点的突变检出率及重复率均达100%。结论:本方法可准确、高通量、快速检测CYP2C9*3突变,特别适宜该SNP位点批量检测需要。

AIM： To establish a pyrosequencingbased method for simultaneous and rapid detection of CYP2C9^＊3 gene polymorphism. METHODS： With the results of standard ABI sequencing as a standard control, we observed and analyzed the reliability of the pyrosequencing method which involves the establishment of biotin-labeled primers, amplification of PCR, separation of Beads, preparation of pyrosequencing single-stranded template of mutational sites and sequencing of PyroMark ID in pymsequencing apparatus by detected 220 cases of human DNA samples. RESULTS： We achieved the simultaneous and rapid detection platform of CYP2C9^＊3 mutation of DNA samples based on Pyrosequencing technology, which can detect DNA samples with high-flux manner. The test results of the CYP2C9^＊3 mutation of 96 DNA samples can be obtained each time with this detection platform. Compared with the standard ABI sequencing, the mutation detection rate and the repetition rate of CYP2C9^＊3 mutation are 100% in this method. CONCLUSION： Pyrosequencing method is a accurate, high-throughput, rapid method for the CYP2C9^＊3 mutation detection, especially suitable for the bulk detection of this mutation.