Miller D N, Bryant J E, Madsen E L, Ghiorse W C
Section of Microbiology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853-8101, USA.
Appl Environ Microbiol. 1999 Nov;65(11):4715-24. doi: 10.1128/AEM.65.11.4715-4724.1999.
We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.
我们使用了两种不同有机质含量的粉质壤土和一种粉质壤土湿地沉积物的冷冻及干燥样本，比较并统计评估了九种DNA提取方法的有效性。基于回收DNA的产量和分子大小，评估了不同化学提取剂（十二烷基硫酸钠[SDS]、氯仿、苯酚、Chelex 100和异硫氰酸胍）、不同物理破碎方法（珠磨均质化和冻融裂解）以及溶菌酶消化的效果。对这九种提取方法的两两比较显示，SDS与氯仿或苯酚联合使用的珠磨均质化优化了提取的DNA量和DNA的分子大小（最大大小为16至20 kb）。在SDS处理前进行溶菌酶消化、异硫氰酸胍处理或添加Chelex 100树脂均未提高DNA产量。当以DNA产量和细胞裂解效率为标准时，发现在含有氯仿、SDS、氯化钠和磷酸盐 - Tris缓冲液（pH 8）的裂解混合物中进行珠磨均质化是最佳的物理裂解技术。还使用两种不同的均质器对珠磨均质化条件的速度和持续时间进行了优化。当使用较低速度和较短时间（30至120秒）时，高分子量DNA的回收率最高。我们评估了四种不同的DNA纯化方法（基于硅胶的DNA结合、琼脂糖凝胶电泳、醋酸铵沉淀和Sephadex G - 200凝胶过滤）用于从粗提物中回收DNA和去除PCR抑制剂。发现Sephadex G - 200旋转柱纯化是去除PCR抑制物质同时使纯化过程中DNA损失最小化的最佳方法。我们的结果表明，对于这些类型的样本，最佳的DNA回收需要在磷酸盐缓冲的SDS - 氯仿混合物存在下进行简短、低速的珠磨均质化，然后进行Sephadex G - 200柱纯化。
