NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE).
Microbes Environ. 2008;23(2):159-66. doi: 10.1264/jsme2.23.159.
A series of additives were evaluated for their effects on improving the yield and quality of DNA extracted from recalcitrant soils. Levels of possible DNA contaminants in these supplements were also assessed. Three of the additives (skim milk, casein, and RNA) were shown to be effective in improving the stable extraction of DNA from recalcitrant samples of Andosol. However, whereas skim milk appeared to be the most effective additive for this purpose, our data indicated that this commercially sourced product contained considerable amounts of contaminant DNA (30 to 40 μg/g skim milk). A ribosomal intergenic spacer analysis (RISA) revealed the consistent contamination of different batches of this product with DNA of several species of both eukaryotes (cattle and protists) and prokaryotes. In particular, thermophilic bacteria such as Geobacillus and Anoxybacillus were identified in the sequenced PCR amplicons from skim milk. The results of the RISA clearly also indicated that the impact of contaminated DNA on the analysis of a microbial community could be significant when skim milk is used for extracting DNA from a recalcitrant soil. In contrast, only a trace amount of contaminating DNA was evident in casein and none was detected in the RNA examined in the present study.
一系列添加剂被评估其对改善从顽固土壤中提取 DNA 的产量和质量的效果。还评估了这些添加剂中可能存在的 DNA 污染物的水平。结果表明，三种添加剂（脱脂乳、酪蛋白和 RNA）可有效提高从顽抗性土壤样本中稳定提取 DNA 的能力。然而，虽然脱脂乳似乎是最有效的添加剂，但我们的数据表明，这种市售产品含有相当数量的污染 DNA（30 至 40μg/g 脱脂乳）。核糖体基因间隔区分析（RISA）显示，不同批次的该产品持续受到真核生物（牛和原生生物）和原核生物多个物种的 DNA 的污染。特别是，在从脱脂乳中提取的 PCR 扩增子的测序中鉴定出了嗜热细菌，如 Geobacillus 和 Anoxybacillus。RISA 的结果还清楚地表明，当从顽固土壤中提取 DNA 时，如果使用脱脂乳，污染 DNA 对微生物群落分析的影响可能是显著的。相比之下，在本研究中，仅在酪蛋白中检测到痕量的污染 DNA，而在检查的 RNA 中则未检测到。
