Assessing the bias linked to DNA recovery from biofiltration woodchips for microbial community investigation by fingerprinting
Assessing the bias linked to DNA recovery from biofiltration woodchips for microbial community investigation by fingerprinting
复制标题
DOI：
10.1007/s00253-009-2253-8
复制
发表时间：
2010-01-01
影响因子：
5
通讯作者：
Fanlo, Jean-Louis
中科院分区：
文献类型：
--
作者：
Cabrol, Lea;Malhautier, Luc;Fanlo, Jean-Louis
在这项研究中，我们探讨了方法学方面的核酸回收的微生物群落参与的气体生物过滤器充满松树皮木屑。通过两个步骤间接回收DNA，比较不同的方法：细胞分散（破碎、振荡和超声处理）和DNA提取（三种商业试剂盒和实验室方案）。目的是（a）优化细胞从填充材料的解吸，和（B）根据三个相关标准比较解吸和提取方法的12种组合：DNA产率、DNA纯度和通过变性梯度凝胶电泳（DGGE）的群落结构表示。细胞分散不受振荡和混合测试的操作参数的影响，而随着超声处理时间的增加。DNA提取的实验室协议提供了最高的DNA产量，而最好的DNA纯度是由一个商业试剂盒，设计用于从土壤中提取DNA。在成功的PCR扩增后，12种方法在微生物群落代表性方面没有产生相同的偏倚。八个组合导致高多样性估计，独立的实验程序。其中，六个提供了高度相似的DGGE配置文件。两种协议产生了显着不同的社区概况，多样性较小。这项研究强调了DNA回收偏倚评估的重要性。
In this study, we explored methodological aspects of nucleic acid recovery from microbial communities involved in a gas biofilter filled with pine bark woodchips. DNA was recovered indirectly in two steps, comparing different methods: cell dispersion (crushing, shaking, and sonication) and DNA extraction (three commercial kits and a laboratory protocol). The objectives were (a) to optimize cell desorption from the packing material and (b) to compare the 12 combinations of desorption and extraction methods, according to three relevant criteria: DNA yield, DNA purity, and community structure representation by denaturing gradient gel electrophoresis (DGGE). Cell dispersion was not influenced by the operational parameters tested for shaking and blending, while it increased with time for sonication. DNA extraction by the laboratory protocol provided the highest DNA yields, whereas the best DNA purity was obtained by a commercial kit designed for DNA extraction from soil. After successful PCR amplification, the 12 methods did not generate the same bias in microbial community representation. Eight combinations led to high diversity estimation, independently of the experimental procedure. Among them, six provided highly similar DGGE profiles. Two protocols generated a significantly dissimilar community profile, with less diversity. This study highlighted the crucial importance of DNA recovery bias evaluation.