用于SSR分析的大豆干种子DNA提取条件的优化

Optimization of the Conditions for Extracting DNA for SSR Analysis from Dry Soybean Seeds

[目的]探讨适于SSR分析的大豆干种子DNA提取的最佳方法。[方法]以2个大豆品种的干种子为材料,分别采用改良SDS法和CTAB法提取大豆基因组DNA,用1%琼脂糖凝胶电泳检测DNA质量,用紫外分光光度计分别测定其DNA在A230、A260和A280下的吸光值,根据A260/A230和A260/A280的比值检测DNA的纯度和浓度,并分别以两种DNA为模版进行SSR-PCR扩增。[结果]相同DNA提取条件下,SDS法提取DNA样品的纯度和浓度均高于CTAB法,SDS终浓度为2%(W/V),水浴15 min,氯仿/异戊醇抽提2次时所提取大豆种子DNA的纯度及浓度最高,其DNA的OD260/OD280值为1.86,SDS法所提的DNA泳带分布较集中,无拖尾现象,其DNA浓度能够满足SSR扩增模板的需求。[结论]SDS浓度为2%(W/V),水浴15 min,氯仿/异戊醇抽提2次时提取大豆干种子DNA的浓度和纯度都较高,可以满足后续的各种分子生物学操作的要求。

[ Objective ] The purpose was to discuss the best way to extract DNA from dry seeds of soybean for SSR analysis. [ Method ] With dry seeds of soybean as tested materials, genomic DNA of soybean was extracted with modified SDS method and CTAB method and examined with 1 % agarose gel electrophoresis. The absorbance value was measured by ultraviolet spectrophotometer at A230 、A260 and A280 and the ratio of A260/A230 and A260/A280 was used to determine the DNA purity and the mass concentration. DNA extracted with 2 methods was used as the template for SSR-PCR amplification. [ Result] The DNA purity and concentration of samples extracted with SDS method were higher than the CTAB method under the same conditions. The SDS conen, of 2% （ W/V）, water bath for 15 min and extracted 2 times with chloroform/ isoamyl alcohol, the DNA purity and concentration exlraeted from soybean seed were the highest and the ratio of OD260/OD280 was 1.86. DNA bands extracted with SDS method were centralized distribution and non-tailing phenomenon which could meet the requirements of SSR amplification template. [ Conclusion] The SDS concn, of 2% （ W/V）, water bath for 15 min, extracted 2 times with chloroform/isoamyl alcohol, the ratio of OD260/OD280 was 1.86, which could meet the requirements of molecular biology research.