A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature

作者：C.L. Moyer, James M. Tiedje, Fred C. Dobbs, David M. Karl · 发表于：Applied and Environmental Microbiology · 年份：1996 · DOI：10.1128/aem.62.7.2501-2507.1996 · 被引用次数：222 · 研究领域：Genomics and Phylogenetic Studies、Microbial Community Ecology and Physiology、Oral microbiology and periodontitis research

An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type and number of TREs has not been systematically evaluated. Our objective was to identify the optimal combination of TREs for RFLP screening of cloned SSU rRNA genes from undefined bacterial clone libraries. After computer-simulated TRE digestion, the resultant fragments were categorized on the basis of the frequency of different restriction fragment size classes. Three groups of distribution patterns for the TREs were determined and further examined via graphical exploratory data analysis. The RFLP size-frequency distribution data for each group of enzymes were then used to infer phylogenetic relationships via the neighbor-joining method. The resulting bootstrap values and the correct placement of node bifurcations were used as additional criteria to evaluate the efficacy of the selected TREs. These RFLP data were compared with known phylogenetic relationships based on SSU rRNA sequence analysis as defined by the Ribosomal Database Project. A heuristic approach testing random combinations of TREs showed that three or more TRE combinatio...