PCR-RFLP方法检测锰超氧化物歧化酶基因V(16)A多态性的实验条件研究

The Study of Conditions Influencing Polymerase Chain Reaction-restriction Fragment Length Polymorphism in the Site of Manganese Superoxide Dismutase Gene V(16)A Polymorphism

目的:确定PCR-RFLP技术检测锰超氧化物歧化酶基因(MnSOD)V(16)A多态性的最适实验条件.方法:对影响MnSOD基因PCR反应以及用限制性内切酶BsawI切割主要因素进行了系统研究.结果:最适退火温度为55℃;最适引物浓度为0.25μmol/L;最适Mg2+浓度为1.5 mmol/L;最适dNTP浓度为0.20 mmol/L;以0.6 U为最适Taq酶量.酶切体系为15μL体系中加8μL产物用2 U的酶消化,为后续研究奠定了实验基础.

Objective： To confirm the optimal test condition of the polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） in the site of manganese superoxide dismutase （MnSOD） gene V（16）A polymorphism. Methods： The factors influencing PCR-RFLP in the site of MnSOD gene V（16）A polymorphism were studied. Results： The results revealed the optimum conditions： annealing temperature was 55℃ ; primer concentration was 0.25 μmol/L; magnesium concentration was 1.5 rnmol/L; concentration of dNTP was 0.20 mmol/L; magnesium concentration was 1.5mmol/L; concentration of dNTP was 0.20 mmol/L; Taq enzyme was 0.6 U; the effective digestion system was 15μL including 8μL DNA solution and 2U enzyme. The information in this study may be useful to relative experiments.