虎杖ISSR-PCR反应体系的建立与优化

An Optimal ISSR-PCR Reaction System for Polygonum cuspidatum

采用SDS法和CTAB法提取虎杖的DNA,并比较两种方法的效果,通过单因子试验优化影响虎杖ISSR-PCR的主要参数。结果表明,CTAB法提取的虎杖总DNA质量优于SDS法。虎杖ISSR-PCR的最佳反应体系总体积为25μL,1×PCRreaction buffer[10mmolTris-HCl(pH8.3),50mmolKCl],40ng模板DNA,0.25mmol·L-1dNTPs,2UTaqDNA聚合酶,2.0mmol·L-1MgCl2,1.0μmol·L-1引物。该反应体系适用于应用ISSR标记开展虎杖DNA指纹、遗传多样性等研究。

The genomic DNA of Polygonum cuspidatum was extracted by SDS and CTAB methods respectively. The effects of ISSR-PCR was analyzed and optimized with single factor experiment. According to the quality of extracted DNA,the CTAB method was better than SDS method. The optimized reaction system was as follows： 25 μL PCR reaction system containing 1xPCR reaction buffer [10mmol Tris-HC1 （pH8.3）, 50 mmol KCl],40 ng template DNA, 0.25 mmol· L^-1 dNTPs, 2U Taq DNA polymerase,2.0 mmol·L^-1 MgCl2 and 1.0 μmol· L^-1 primer. The ISSR-PCR system is suitable for the study of the Polygonum cuspidatum DNA fingerprint, genetic diversity, etc.