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从消化物和粪便样本中改进提取用于聚合酶链反应(PCR)的高质量群落DNA。

Improved extraction of PCR-quality community DNA from digesta and fecal samples.

作者信息

Yu Zhongtang, Morrison Mark

机构信息

Dept. of Animal Sciences, Ohio State University, 2027 Coffey Road, OH 43210, USA.

出版信息

Biotechniques. 2004 May;36(5):808-12. doi: 10.2144/04365ST04.

Abstract

Several DNA extraction methods have been reported for use with digesta or fecal samples, but problems are often encountered in terms of relatively low DNA yields and/or recovering DNA free of inhibitory substances. Here we report a modified method to extract PCR-quality microbial community DNA from these types of samples, which employs bead beating in the presence of high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA, and with subsequent DNA purification by QIAamp columns [referred to as repeated bead beating plus column (RBB + C) method]. The RBB + C method resulted in a 1.5- to 6-fold increase in DNA yield when compared to three other widely used methods. The community DNA prepared with the RBB + C method was also free of inhibitory substances and resulted in improved denaturing gradient gel electrophoresis (DGGE) profiles, which is indicative of a more complete lysis and representation of microbial diversity present in such samples.

摘要

已有多种DNA提取方法被报道可用于消化物或粪便样本,但在DNA产量相对较低和/或回收不含抑制性物质的DNA方面,常常会遇到问题。在此,我们报告一种改良方法,用于从这类样本中提取PCR级别的微生物群落DNA,该方法在高浓度十二烷基硫酸钠(SDS)、盐和乙二胺四乙酸(EDTA)存在的情况下进行珠磨,随后通过QIAamp柱进行DNA纯化[称为重复珠磨加柱(RBB + C)法]。与其他三种广泛使用的方法相比,RBB + C法使DNA产量提高了1.5至6倍。用RBB + C法制备的群落DNA也不含抑制性物质,并使变性梯度凝胶电泳(DGGE)图谱得到改善,这表明此类样本中的微生物多样性得到了更完全的裂解和呈现。

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