Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses
Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses
复制标题
从浮游细菌中同时提取总 RNA 和 DNA，适合定量结构和功能分析
DOI：
10.1128/aem.68.3.1082-1087.2002
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发表时间：
2002
影响因子：
4.4
通讯作者：
M. Höfle
中科院分区：
文献类型：
--
作者：
M. Weinbauer;I. Fritz;D. Wenderoth;M. Höfle
摘要：本研究旨在建立一种同时从浮游细菌中提取RNA和DNA用于定量分子分析的方法。采用机械和化学相结合的提取方法，以十二烷基肌酸钠-苯酚或双伏拉布-苯酚为提取混合物，RNA和DNA得率最高。革兰氏阴性分离菌和浮游细菌的核酸提取效率相对较高，差异不大（RNA为52 ~ 66%，DNA为43 ~ 61%）；从革兰氏阳性细菌分离物中也获得了大量的核酸（RNA, 20 - 30%； DNA, 20 - 25%）。从所有分离物和群落中获得16S rRNA及其基因片段的逆转录PCR和PCR扩增产物，表明提取的核酸完整且纯度足以用于群落结构分析。利用16S rRNA及其基因片段的单链构象多态性，获得了池塘浮游细菌的群落指纹图谱。在池塘水样中可检测到亚硝酸盐还原酶基因（nir基因）片段的mRNA转录本，表明该提取方法也适用于研究基因表达。所提出的提取方法产生的核酸可用于从单个样品中进行浮游细菌群落的结构和功能研究。
ABSTRACT The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (nir gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample.