Evaluation of methods for the extraction and purification of DNA from the human microbiome.
Evaluation of methods for the extraction and purification of DNA from the human microbiome.
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DOI：
10.1371/journal.pone.0033865
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Forney LJ
中科院分区：
文献类型：
--
作者：
Yuan S;Cohen DB;Ravel J;Abdo Z;Forney LJ
DNA提取是所有与培养无关的方法中的关键步骤，以表征微生物多样性，包括与人体相关的微生物多样性。使用这些方法的一个根本挑战是分离出代表样本微生物群落的DNA。在这项研究中，我们对六种常用的DNA提取程序进行了统计评估，使用了11种与人类相关的细菌物种和一个包含相同数量的这11种细菌的模拟群落。这些方法在DNA产量、DNA剪切、重复性以及最重要的代表微生物多样性的基础上进行了比较。对模拟群落的16S rRNA基因序列分析表明，所用的6种DNA提取方法所观察到的物种丰富度与预期物种丰富度有显著差异。包括打珠和/或致突变素的方案比不包括这两种方案的方法产生显著更好的细菌群落结构表示。6种方法的重复性相似，不同实验者、不同时间的结果吻合较好。根据所做的评估，似乎用于人类相关样本细菌群落分析的DNA提取程序应该包括打珠和/或突变素，以有效地裂解细胞。
DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.
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