Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes
Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes
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DOI：
10.1128/aem.68.7.3243-3250.2002
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发表时间：
2002-07
影响因子：
4.4
通讯作者：
L. Valinsky;G. Della Vedova;A. Scupham;S. Alvey;A. Figueroa;Bei Yin;R. J. Hartin;M. Chrobak;D. Crowley;T. Jiang;J. Borneman
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文献类型：
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作者：
L. Valinsky;G. Della Vedova;A. Scupham;S. Alvey;A. Figueroa;Bei Yin;R. J. Hartin;M. Chrobak;D. Crowley;T. Jiang;J. Borneman
摘要 描述生态系统特征的第一步是描述居住在其中的生物体。对于微生物研究来说，实验的局限性阻碍了描绘多样化群落的能力。在这里，我们描述了 rRNA 基因的寡核苷酸指纹图谱 (OFRG)，这种方法允许通过使用小 DNA 探针的一系列杂交实验来识别阵列的 rRNA 基因 (rDNA)。为了证明这一策略，我们检查了两种不同土壤中的细菌。对 1,536 个 rDNA 克隆的分析揭示了 766 个簇，分为五个主要分类群：芽孢杆菌、放线菌、变形菌和两个未定义的组合。土壤特异性分类群被鉴定出来，然后通过原始土壤 DNA 的簇特异性 PCR 进行独立确认。通过该分析获得了接近物种水平的分辨率，因为平均序列同一性为 97% 的克隆被分组在同一簇中。将这些 OFRG 结果与相同两种土壤的变性梯度凝胶电泳分析结果进行比较，证明了这一方法进步的重要性。 OFRG 提供了一种经济有效的方法来广泛分析微生物群落，并且应该在医学、生物技术和生态系统研究中得到应用。
ABSTRACT One of the first steps in characterizing an ecosystem is to describe the organisms inhabiting it. For microbial studies, experimental limitations have hindered the ability to depict diverse communities. Here we describe oligonucleotide fingerprinting of rRNA genes (OFRG), a method that permits identification of arrayed rRNA genes (rDNA) through a series of hybridization experiments using small DNA probes. To demonstrate this strategy, we examined the bacteria inhabiting two different soils. Analysis of 1,536 rDNA clones revealed 766 clusters grouped into five major taxa: Bacillus, Actinobacteria, Proteobacteria, and two undefined assemblages. Soil-specific taxa were identified and then independently confirmed through cluster-specific PCR of the original soil DNA. Near-species-level resolution was obtained by this analysis as clones with average sequence identities of 97% were grouped in the same cluster. A comparison of these OFRG results with the results obtained in a denaturing gradient gel electrophoresis analysis of the same two soils demonstrated the significance of this methodological advance. OFRG provides a cost-effective means to extensively analyze microbial communities and should have applications in medicine, biotechnology, and ecosystem studies.