风沙土微生物总DNA不同提取和纯化方法比较

Comparison of the methods for extracting and purifying microbial total DNA from an aeolian sandy soil

为筛选和建立风沙土中总DNA的提取和纯化方法,选取了5种直接提取法、1种间接提取法和2种纯化法分别对风沙土中总DNA进行了提取和纯化,并对其质量和产量进行了比较.结果表明:6种方法均可从风沙土中提取到大小为23kb左右的总DNA,其中改进后的高盐提取法(用40%聚乙二醇8000和4mol·L-1NaCl沉淀DNA)效果最好,纯化后总DNA的纯度最高,可进行16S rDNA的PCR扩增,且产量仅稍低于试剂盒提取法;电泳加柱回收纯化法的纯化效果较好,经该方法纯化后的总DNA大部分可进行PCR扩增,可满足后续分子操作对DNA纯度的要求.

In order to select and establish an appropriate method for extracting and purifying the microbial total DNA from an aeolian sandy soil,six extraction methods （five direct methods and one indirect method） and two purification methods were examined,with the quantity and quality of extracted and purified total DNA compared. All the six extraction methods could extract the total DNA with a length of approximately 23 kb,among which,the improved SDS high salt extraction method （using 40% PEG8000 and 4 mol·L-1 NaCl to precipitate DNA） was the best. This method could have a yield slightly less than that obtained by using kits,and the extracted DNA had the highest purity after purification,being available in 16S rDNA PCR amplification. Among the purification methods,the effect of agarose gel electrophoresis plus minicolumn was satisfactory,with most of the purified total DNA being able to be PCR-amplified and meet the requirements of the purity of DNA in the follow-up molecular operations.