大豆叶片DNA提取方法的比较研究

COMPARATIVE STUDIES ON EXTRACTION OF GENOME DNA FROM SOYBEAN LEAF

以大豆叶片为材料,对大豆基因组DNA提取方法中若干影响因子如提取液、提取液的浓度、蛋白质去除次数、提取步骤等进行比较研究,试图寻找大豆叶片DNA提取的最佳方法。结果表明,碱裂解法提取速度快,但提取的DNA量少,在SSR分析中,银染效果较CTAB、SDS提取液差,CTAB、SDS提取液均能得到较高质量的DNA;在1%-4%CTAB、SDS提取液浓度下,4%CTAB易使DNA产生降解,但不同浓度提取液提取DNA均可用于SSR分析;提取叶片重量在一定范围内时,随抽提次数减少,DNA浓度增加,纯度下降,但即使用氯仿/异戊醇抽提一次,也能够满足SSR分析的需要;不同提取步骤下,使用氯仿/异戊醇抽提2次后使DNA沉出后再溶解抽提2次和直接抽体4次相比,A260/A280值偏大,但A260/A230值较好。

Template DNA quality directly affects the effectiveness of PCR amplification. To investigate the optimum DNA extraction method from soybean leaf, the influencing factors such as extraction buffer, buffer concentration, extraction times, and extraction steps were analyzed. The results indicated that the alkali extraction method on leaf was faster than but inferior to the other two methods. Both CTAB and SDS method could produce high quality DNA. When the DNA quality was compared under different buffer concentration ranging from 1% to 4%, it was found the quality of DNA could meet need of SSR analysis except that the DNA degraded a little under 4% CTAB. The concentration of DNA increased as the extraction times decreased, while the purity of DNA descended. The quality of DNA could meet SSR requirement even extracted once by chloroform/isoamyl. Under different extraction steps, we extracted DNA 4 times continuously, or separated out DNA after extracting twice, then dissolved it and extracted twice more, compared to the former, the later had a bigger ratio of A260/A280, but a suitable ratio of A260/ A230.