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  • terminal Restriction Fragment length polymorphism analysis program a web based research tool for microbial community analysis
    Applied and Environmental Microbiology, 2000
    Co-Authors: Terence L. Marsh, Paul Saxman, James R Cole, James M Tiedje
    Abstract:

    Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses.html) that facilitates microbial community analysis using terminal Restriction Fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico Restriction digestions of the entire 16S sequence database and derive terminal Restriction Fragment sizes, measured in base pairs, from the 5′ terminus of the user-specified primer to the 3′ terminus of the Restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal Restriction Fragment length polymorphisms.

  • characterization of microbial diversity by determining terminal Restriction Fragment length polymorphisms of genes encoding 16s rrna
    Applied and Environmental Microbiology, 1997
    Co-Authors: Wen Tso Liu, Terence L. Marsh, Hans H Cheng, Larry J Forney
    Abstract:

    A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5* end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with Restriction enzymes, and the fluorescently labeled terminal Restriction Fragment was precisely measured by using an automated DNA sequencer. Computersimulated analysis of terminal Restriction Fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and Restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal Restriction Fragment lengths or “ribotypes.” Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.