Steffan R J, Goksøyr J, Bej A K, Atlas R M
Department of Biology, University of Louisville, Kentucky 40292.
Appl Environ Microbiol. 1988 Dec;54(12):2908-15. doi: 10.1128/aem.54.12.2908-2915.1988.
Experiments were performed to evaluate the effectiveness of two different methodological approaches for recovering DNA from soil and sediment bacterial communities: cell extraction followed by lysis and DNA recovery (cell extraction method) versus direct cell lysis and alkaline extraction to recover DNA (direct lysis method). Efficiency of DNA recovery by each method was determined by spectrophotometric absorbance and using a tritiated thymidine tracer. With both procedures, the use of polyvinylpolypyrrolidone was important for the removal of humic compounds to improve the purity of the recovered DNA; without extensive purification, various restriction enzymes failed to cut added target DNA. Milligram quantities of high-purity DNA were recovered from 100-g samples of both soils and sediments by the direct lysis method, which was a greater than 1-order-of-magnitude-higher yield than by the cell extraction method. The ratio of labeled thymidine to total DNA, however, was higher in the DNA recovered by the cell extraction method. than by the direct lysis method, suggesting that the DNA recovered by the cell extraction method came primarily from active bacterial cells, whereas that recovered by the direct lysis method may have contained DNA from other sources.
开展了实验以评估两种不同方法从土壤和沉积物细菌群落中回收DNA的有效性：先进行细胞提取，然后进行裂解和DNA回收（细胞提取法），以及直接细胞裂解和碱性提取以回收DNA（直接裂解法）。通过分光光度吸光度并使用氚化胸腺嘧啶示踪剂来确定每种方法回收DNA的效率。对于这两种方法，使用聚乙烯聚吡咯烷酮对于去除腐殖化合物以提高回收DNA的纯度很重要；如果不进行广泛纯化，各种限制酶无法切割添加的目标DNA。通过直接裂解法从100克土壤和沉积物样品中回收了毫克量的高纯度DNA，其产量比细胞提取法高出一个多数量级。然而，细胞提取法回收的DNA中标记胸腺嘧啶与总DNA的比率高于直接裂解法。这表明细胞提取法回收的DNA主要来自活性细菌细胞，而直接裂解法回收的DNA可能包含来自其他来源的DNA。
