2. T-RFLP Methodology and Web Site
Over the past century we have learned that microbial communities have enormous phylogenetic and physiological complexity. It is only within the past 25 years that we have begun to tease apart the phylogenetic relationships with comparative nucleic acid and protein sequence analysis. Ribosomal RNA, specifically 16S rRNA, has turned out to be the premier phylogenetic molecule for these comparative studies while PCR amplification of this template from total community DNA has allowed us to view beyond the cultivable members of communities. Using these approaches, we have detected hundreds of communities comprised of thousands of "species" or distinct taxonomic units. Clearly an approach that systematically compares and contrasts these communities would significantly advance our understanding of the structure and function of the microbial world. However, because of the complexity of communities, comparative community analyses based solely on sequencing is not financially possible. For this reason, other approaches that provide a means to rapidly screen communities using phylogenetic markers such as 16S, but applying novel separation techniques to the PCR amplified gene, have been developed. At the Center for Microbial Ecology we have helped to develop a community analysis approach that uses the size of terminal restriction fragments from amplification products as the initial comparative tool. An example of the analysis of a community using this technique for three different genes is illustrated in Figure 1. In this approach the target gene is amplified from the community using standard PCR techniques with a 5' fluorescently tagged primer. The amplification products are digested with a restriction endonuclease and run on an automated sequencer. Because only the restriction fragment proximal to the labeled primer carries the fluor, only this fragment is detected by the automated system. In general, each population of the community contributes a terminal fragment of one size. Because the analysis is based on the size, in basepairs, of the terminal fragments, direct comparisons can be made with the large sequence database of ribosomal RNA (Ribosomal Database Project). To facilitate this comparison, we have developed a web site that permits on line in silico restriction digestion of the current release of aligned sequences from the RDP (see http://rdp.cme.msu.edu/html/ for T-RFLP tools). The size of the terminal fragments from restriction digests with operator-selected primer sequences and enzyme(s) can be displayed phylogenetically or sorted by fragment size. Hence the investigator can determine which primer-enzyme combination is optimal for tracking a known phylogenetic group within a community or which groups are potential members of an unknown community. While one cannot derive phylogeny with one or even several terminal fragments, the information can provide direction to subsequent experiments.
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