Abstract

Microbial populations in complex environmental samples are difficult to characterize; current techniques are incomplete and time consuming. We investigated a polymerase chain reaction (PCR)-based method for rapidly comparing bacterial communities independent of culture or cloning. Community 16S rRNA genes were amplified and fluorescently labeled by PCR. The labeled products were digested by a restriction enzyme and the labeled, terminal restriction fragments (TRFs) were separated by electrophoresis and detected by laser-induced fluorescence on an automated gene sequencer. PCR parameters were optimized using an in vitro model community of known organisms. Community comparisons were made between deer fecal pellets, petroleum hydrocarbon-contaminated sands and pristine sand. Principal components analysis (PCA) was used to compare the resulting TRF patterns (TRFPs). Patterns derived from a single enzyme digest did not result in accurate community characterizations. Accurate characterizations reflecting the expected bacterial community biology were only achieved by combining TRFP data derived from different enzyme digestions. Suggestions are offered for future use of this technique.

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Biology

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Biology Commons

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URL: http://digitalcommons.calpoly.edu/bio_fac/70