Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/1997
Publication Date: N/A
Interpretive Summary: The evaluation of genetic diversity is important to know especially when it comes to populations that are constrained by outside forces. In this study, a new technique was developed to measure the amount of genetic diversity in microbial populations. Compared to previous techniques, the T-RFLP technique is more sensitive, quantitative, and relatively fast. This procedure provides a powerful tool to researchers working on genetic diversity and germplasm preservation.
Technical Abstract: A quantitative molecular technique was developed for the characterization of microbial diversity and the composition of communities from various environments. The technique employed PCR wherein one of the two primers used was fluorescently-labeled at the 5**' end and used to amplify a selected region of bacterial 16S rDNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured using an automated DNA sequencer. Computer simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1002 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 gut; as many as 72 unique ribotypes were found among those communities with 36 ribotypes observed in the termite gut. The similarity of community T-RFLP patterns was numerically analyzed with band-based coefficients, and hierarchiaclly clustered. The pattern derived from termite gut was found to be distinctly different 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 community structure in different ecosystems.