CYP2C19基因多态性PCR扩增体系的正交优化与验证

Orthogonal Optimization and Verification of PCR Amplification System for CYP2C19 Gene Polymorphism

本实验通过建立CYP2C19*2、*3和*17基因多态性PCR反应体系和条件,筛选出4μL体系中各因素最佳水平。采用SNaPshot技术对CYP2C19基因3个SNP位点*2、*3和*17同时进行复合扩增检测,利用L9(34)正交实验设计,对影响PCR反应体系和条件的3个因素(PCR Mix, Taq DNA聚合酶,循环次数)在3个水平上进行优化,结果采用综合评分法和极差分析法进行分析。用3组已知样本对正交优化所得条件进行重复性和稳定性验证。结果表明CYP2C19*2、*3和*17基因PCR扩增体系的影响因素依次为:PCR Mix>循环数>Taq DNA聚合酶。最佳反应体系为PCR Mix 2.0μL、Taq DNA聚合酶0.2μL、循环次数32次。3组样本验证效果满意。优化的CYP2C19*2、*3和*17基因PCR反应体系稳定性高,重复性和经济性较好,为该基因多态性的大规模调查奠定了基础。

This study established the PCR reaction system and conditions for investigating of CYP2 C19*2,*3 and*17 gene polymorphisms, and screened the optimal level of each factors in 4 μL system. Three SNPs locus of *2,*3 and*17 in CYP2 C19 gene were applied the complex amplification detection by SNa Pshot method at the same time. Three factors(PCR Mix, Taq DNA polymerase and cycles) influencing the PCR reaction system were optimized with L9(34)orthogonal experimental design on three levels. Then the results were analyzed by methods of comprehensive evaluation and range analysis. Finally, the repeatability and stability of optimized conditions were verified by three groups of known samples. The results showed that the influence factors of the PCR amplification system of CYP2 C19*2,*3 and *17 gene were PCR Mix, then the number of cycles and last the Taq DNA polymerase. Optimal reaction system was 2.0 μL PCR Mix, 0.2 μL Taq DNA polymerase and 32 times of cycles. Three groups of known samples were verified satisfactory. The optimized PCR system of CYP2 C19*2,*3 and *17 gene was testified with high stability and better repeatability and economy, which would lay the foundation for large-scale investigation of the gene polymorphism.