Department of Environmental Sciences and Engineering, The University of North Carolina at Chapel Hill, CB #7431, Chapel Hill, North Carolina 27599-7431, USA.
Appl Environ Microbiol. 2011 May;77(9):2984-91. doi: 10.1128/AEM.01942-10. Epub 2011 Mar 11.
In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the (13)C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-(13)C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from (13)C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.
在迄今为止发表的许多基于 DNA 的稳定同位素探测 (SIP) 研究中，对土壤样本进行了一次 DNA 提取，通常使用商业 DNA 提取试剂盒，然后通过密度梯度超速离心回收（13）C 标记的（重）DNA。然而，最近的证据表明，对土壤样本进行一次提取可能无法代表样本中所有生物体的代表性回收。为了确定多次 DNA 提取是否会影响 DNA 产量、细菌 16S rRNA 基因拷贝数或蒽降解细菌的鉴定，我们对相同的受污染土壤样本进行了七次连续的 DNA 提取，这些样本未经处理或用 [U-(13)C]蒽富集。从未处理和蒽富集土壤样本中最大限度地提高 DNA 产量和 16S rRNA 基因拷贝数需要进行多次提取。在（13）C 富集 DNA 衍生的 16S rRNA 基因克隆文库中，优势度顺序为单胞菌目，但与任何先前描述的属都没有关系的序列被命名为“蒽组 1”。与丰度较高的鞘氨醇单胞菌属聚类的序列以及与 Pigmentiphaga 属相关的序列与蒽降解新相关。在所有七次提取中共同鉴定的细菌群都在第一次提取中回收，尽管对 SIP 鉴定的群体进行定量 PCR 分析表明提取模式存在定量差异。这些结果表明，对土壤样本进行多次 DNA 提取可提高可提取的 DNA 产量和可定量的细菌 16S rRNA 基因拷贝数，但对通过 SIP 鉴定与特定碳源降解相关的细菌群的鉴定几乎没有定性影响。
