Isolation of total microbial DNA with high purity is the basis for soil metagenomic study, and it is also the key to analyze the influence of biochar on soil microbial community structure. Because of the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is more difficult than the extraction of DNA from other environmental samples. In order to find a suitable method to extract DNA from biochar-amended soil, 5 different methods were used, including E.Z.N.A. Soil DNA Kit method (method 1), modified E.Z.N. A. Soil DNA Kit method (method 2), SDS-glass beads method (method 3), Mo-Bio Power Soil DNA Isolation Kit method (method 4) and modified Mo-Bio Power Soil DNA Isolation Kit method (method 5). The extracted DNA was subjected to agarose gel electrophoresis analysis and its concentration and purity were determined. The results showed that all of the 5 methods could obtain the soil DNA. The size of DNA was uniform, and the band was relatively simple. At the same time, the concentrations of DNA extracted from the two mixed soil samples were from 3.22 μg·g-1 dry soil to 37.80 μg·g-1 dry soil. The DNA extraction of soil sample 1 was significantly larger than that of soil sample 2. However, there were significant differences in the yield and quality of DNA extracted by different methods. Among them, the DNA yield of method 3 was the highest, the two soil samples were 37.80 μg·g-1 dry soil and 10.78 μg·g-1 dry soil respectively. But its DNA purity was the lowest. The A260/A230 values of the two soil samples were only 0.664 and 0.684, and the A260/A280 values were also less than 1.6, indicating that there was serious contamination of protein and humic acid in the extracted products and they could not be directly used for PCR amplification. Although the DNA yield of method 2 was relatively lower, its DNA purity was the highest. The A260/A280 values of the two soil samples were 1.854 and 1.844, respectively, and the A260/A230 values were 1.857 and 1.663, respectively, which indicated that the removal of protein and humic acid was more thorough. Because method 2 was improved on the basis of a commercial kit, it was simple, time-saving, and reproducible. Based on the results obtained in the study, the modified E.Z.N.A. Soil DNA Kit method could obtain the highest quantity of soil DNA, and it is an ideal method for extracting DNA from biochar-amended soil to meet the requirement of constructing metagenomic library.%获得高纯度的总DNA是进行土壤微生物宏基因组学研究的基础,也是分析生物炭作为土壤改良剂对土壤菌群结构影响的关键.由于生物炭对DNA中磷酸骨架的吸附作用,从添加生物炭的土壤中提取总DNA较为困难.为了寻找合适的提取混有生物炭的土壤总DNA的方法,本研究分别采用E.Z.N.A.Soil DNA Kit法(方法1)、改良的E.Z.N.A.Soil DNA Kit法(方法2)、SDS-玻璃珠法(方法3)、Mo-Bio Power Soil DNA Isolation Kit法(方法4)和改良的Mo-Bio Power Soil DNA Isolation Kit法(方法5)对两种类型的混有生物炭的土壤样品进行总DNA的提取,并对提取的DNA进行琼脂糖凝胶电泳分析以及浓度和纯度的测定.结果表明:5种方法均可从土壤中提取到DNA,并且DNA大小一致,条带也比较单一.同时,采用不同的方法从两种混合土样中提取的DNA浓度范围为3.22～37.80μg·g-1干土,且土样1的DNA提取量明显大于土样2的DNA提取量.但是不同方法提取到DNA的产量和质量存在明显差异,其中方法3提取的DNA得率最高,两种土样分别为37.80μg·g-1干土和10.78μg·g-1干土,但是该方法提取的DNA纯度最低,两种土样DNA的A260/A230值仅为0.664和0.684,A260/A280值小于1.6,说明提取产物中存在严重的蛋白质和腐殖酸的污染,不能直接用于PCR扩增.而方法2虽然提取的DNA得率相对较低,但纯度最高,两种土样的A260/A280值分别为1.854和1.844,A260/A230值分别为1.857和1.663,说明蛋白质和腐殖酸的去除较彻底,并且该方法是在商品化的试剂盒基础上进行的改进,操作简单、省时,重复性好.这些研究结果证明方法2即改良的E.Z.N.A.Soil DNA Kit提取方法,提取到的DNA纯度最高,是较为理想的混有生物炭土壤的DNA提取方法,可以满足构建宏基因组文库的要求.
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