葡萄柚基因组DNA提取方法的比较及ISSR-PCR体系的优化

Comparison of Genomic DNA Extraction Method and Optimization of ISSR-PCR Amplification for Grapefruit

对4种基因组DNA提取方法进行比较,得到一种效率较高的、且适用于葡萄柚ISSR-PCR的DNA提取方法。同时,采用正交设计对影响葡萄柚ISSR-PCR的因子进行优化。实验结果表明,改良的SDS法提取的基因组DNA最适宜进行葡萄柚的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2.7条。葡萄柚的最优ISSR-PCR反应体系为:20μL总体积中含有2×buffer(Mg2+),0.2mmol/L dNTPs,0.2μmol/L引物,0.3U Taq DNA聚合酶,5ng DNA模板。

An efficient genomic DNA extraction method useful for ISSR-PCR of grapefruit was selected by comparing four DNA extraction methods.Meanwhile,the factors influencing ISSR-PCR for grapefruit were optimized by orthogonal design.The results showed that improved SDS method was suitable for ISSR-PCR amplification.The average amplified bands were 2.7 which were detected in 2% agarose.The optimum amplification condition of ISSR-PCR was including 5 ng DNA template,0.2 mmol/L dNTPs,0.2 μmol/L primer,0.3 U Taq DNA polymerase and 2×buffer （Mg 2＋） in 20 μL reaction volumes.