美国白蛾基因组DNA提取及RAPD反应条件优化

Extraction of genomic DNA and optimization of RAPD system for Hyphantria cunea

用改进的SDS法从无水乙醇浸泡美国白蛾幼虫标本中提取到高分子量的可用于RAPD扩增的基因组DNA。用正交试验对影响RAPD扩增的条件进行优化得到最佳反应体系:20μL反应体系中,模板浓度1.25 ng/μL、引物浓度1.2 ng/μL、dNTP浓度0.2 mmol/L、Mg2+浓度3.0 mmol/L、TaqDNA聚合酶用量2.5 U。确定最佳退火温度为38℃。PCR反应条件:94℃30 s、38℃60 s、72℃60 s 35个循环后,72℃延伸5 min。为在分子水平上讨论美国白蛾种群的遗传分化提供基础。

By using improved SDS method, high molecular weight DNAs were extracted from ethanol immersed Hyphantria cunea for RAPD. The factors influencing RAPD reactions were studied and optimized with orthogonal design. The results showed that the optimal conditions were as followed： 1.25 ng/μL of DNA, 1.2 ng/μL of primers, 0. 2 mmol/L of dNTPs, 3.0 nmlol/L of Mg^2＋, 2.5U of Taq DNA polymerase, 2 μL 10×Buffer and 12.6μL ddH2O in a total 20μL RAPD reaction system. The optimized PCR program was： denaturing at 94℃ for 30 s, annealing at 38℃ for 60s, extension at 72℃ for 60 s, for 35 cycles, followed by a final extension at 72℃ for 5 min.