PCR-RLFP方法检测eNOS G894T多态性实验条件研究

Experimental condition research of PCR-RLFP in detection of polymorphism of eNOS G894T

目的探讨聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测内皮细胞型一氧化氮合酶(e NOS)G894T多态性的最佳实验条件。方法对PCR和RFLP的一些影响因素进行研究。结果 PCR扩增e NOS基因的最佳条件为:20μmol/L的引物浓度;5.0 U的Taq酶量;112μmol/L的d NTP浓度。20μl体系中加4μl产物,5.0 U的酶消化4 h为最经济有效的酶切体系。结论最佳的实验条件的探索是进行大批量实验研究的关键。

Objective To investigate the optimum experimental condition of polymerase chain reaction-restricted fragment length polymorphisms （PCR-EFLP） in detection of polymorphism of endothelial nitric oxide synthase （eNOS） G894T. Methods Research was made on influencing factors of PCR and RFLP. Results The optimum experimental conditions for PCR in amplification of eNOS gene included primer concentration as 20μmol/L, Taq enzyme amount as 5.0 U, and dNTP concentration as 112μmol/L. 4μl products in 20μl system under 4 h of 5.0 U enzymic digestion was the most financial enzymatic system. Conclusion Investigation of the optimum experimental condition is the key to implement large quantities of experiments.