拟穴青蟹ISSR-PCR条件的优化

Optimization of Experiment Conditions with ISSR-PCR for Mud Crab Scylla paramamosain

分析DNA模板、Mg2+、dNTPs、引物和Taq DNA聚合酶及甲酰胺、二甲基亚砜等对拟穴青蟹ISSR-PCR反应的影响。研究结果确定了获得清晰条带的模板DNA、Mg2+、dNTPs、引物、Taq DNA聚合酶的浓度范围分别为:模板DNA 8～50 ng/μl,Taq DNA聚合酶0.75～1.0 U,Mg2+1.0～2.0mmol/L(与引物密切相关),dNTP 0.15～0.20 mmol/L,引物浓度0.6～0.8μmol/L。甲酰胺的添加并不能优化拟穴青蟹ISSR-PCR反应结果,而低浓度的二甲基亚砜可提高扩增产物的清晰度,但与引物相关。

The effects of diverse factors on the PCR-ISSR were analyzed for the genetic diversity of Mud Crab Scylla paramamosain. The concentration scope of several factors, i. e. , template DNA, Taq DNA polymerase, Mg2＋ , dNTPs, and Primers were delimited. The addition of the Formamide deteriorated the band type. The Dimethyl Sulfoxide（DMSO）aceompanyed by the special primers could enhance the lighehess of the DNA bands, however, altered the band type, especially in the band placement. Undoubtedly, the optimal ISSR-PCR conditions will be helpful to investigate the population genetic structure of the Mud Crab.