西番莲基因组DNA的提取及ISSR-PCR的优化

Improvement of Genomic DNA Extraction and Optimization of ISSR-PCR Amplification for Passion Fruit

对5种DNA提取方法进行比较,得到一种效率较高的、且适用于西番莲ISSR-PCR的DNA提取方法.同时,对影响西番莲ISSR-PCR的因子进行优化.结果表明:改良的SDS法2提取的DNA最适宜进行西番莲的ISSR-PCR扩增.ISSR-PCR产物在2%琼脂糖凝胶上检测,发现PCR扩增的平均条带数为4.1 2条.西番莲的ISSR-PCR的最优体系为20μL PCR反应液体系中含有1×buffer(Mg2+),0.2 mmol/L dNTPs,0.5μmol/L引物,0.2 UTaqDNA聚合酶,5 ng DNA模板.

By comparing five DNA extraction methods,an efficient method suitable for ISSR-PCR of passion fruit（Passiflora edulis） was identified.The factors influencing ISSR-PCR for passion-fruit were optimized.The result showed that the modified SDS-II procedure was most suitable for ISSR-PCR amplification for passion fruit.Detected on 2% agarose,4.12 amplification bands on the average were observed in the ISSR-PCR products.The optimum amplification conditions for ISSR-PCR of passion fruit were 5 ng DNA template,0.2 mmol/L dNTPs,0.5 μmol/L primer,0.2 U Taq DNA polymerase and 1× buffer（Mg2＋） in 20 μL reaction volumes.