Zhang Xueli, Yan Xing, Gao Pingping, Wang Linghua, Zhou Zhihua, Zhao Liping
Laboratory of Molecular Microbial Ecology and Ecogenomics, College of Life Science and Biotechnology, Shanghai Jiao Tong University, #800 Dongchuan Road, Shanghai 200240, China.
J Microbiol Methods. 2005 Jan;60(1):1-11. doi: 10.1016/j.mimet.2004.08.015.
Sequence retrieval from single bands of polymerase chain reaction (PCR)-denaturing gel electrophoresis (DGE) profiles is an important but often difficult step for molecular diversity analysis of complex microbial communities such as activated sludge systems. We analyzed the temperature gradient gel electrophoresis (TGGE) profiles of PCR-amplified 16S rDNA fragments from an activated sludge sample of a coking wastewater treatment plant. Single bands were excised, and a clone library was constructed for each. Sequence heterogeneity in each single band was found to be significantly overestimated due to single-stranded DNA (ssDNA) contamination formed during the PCR amplification, since only 10-60% of library clones of each single TGGE band had identical migration behavior compared with the parent band. Three methods, digestion with mung bean nuclease, optimization of PCR amplification, and purification via denatured polyacrylamide gel electrophoresis (d-PAGE), were compared for their ability to minimize ssDNA contamination, with the last one being the most efficient. After using d-PAGE to minimize ssDNA to a nearly nondetectable level, 70-100% of library clones for each single TGGE band had identical migration compared with the parent band. Several sequences were found in each of six single bands, and this co-migration could be predicted with the Poland software. The predominant bacteria of the activated sludge were assessed via a combination of sequence retrieval from each single TGGE band and band intensity analysis. Only beta and alpha subclasses of the Proteobacteria were detected, 93.8% and 6.2%, respectively. Our work suggests that prior to constructing a clone library to retrieve the actual sequence diversity of a single DGE band, it is advisable to minimize ssDNA contamination to a nondetectable level.
从聚合酶链反应（PCR）-变性凝胶电泳（DGE）图谱的单一条带中检索序列，对于诸如活性污泥系统等复杂微生物群落的分子多样性分析而言，是重要但往往困难的一步。我们分析了来自某焦化废水处理厂活性污泥样品的PCR扩增16S rDNA片段的温度梯度凝胶电泳（TGGE）图谱。切下单一条带，并为每个条带构建一个克隆文库。发现由于PCR扩增过程中形成的单链DNA（ssDNA）污染，每个单一条带中的序列异质性被显著高估，因为与母带相比，每个单一TGGE条带的文库克隆中只有10%-60%具有相同的迁移行为。比较了三种方法，即绿豆核酸酶消化、PCR扩增优化和变性聚丙烯酰胺凝胶电泳（d-PAGE）纯化，以最小化ssDNA污染的能力，最后一种方法效率最高。在使用d-PAGE将ssDNA最小化到几乎检测不到的水平后，每个单一TGGE条带的文库克隆中有70%-100%与母带具有相同的迁移行为。在六个单一条带中的每一个中都发现了几个序列，并且这种共迁移可以用Poland软件预测。通过从每个单一TGGE条带检索序列并结合条带强度分析，评估活性污泥中的优势细菌。仅检测到变形菌门的β和α亚类，分别为93.8%和6.2%。我们的工作表明，在构建克隆文库以检索单个DGE条带的实际序列多样性之前，最好将ssDNA污染最小化到检测不到的水平。
