Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities

An automated method of ribosomal intergenic spacer analysis (ARISA) was dev eloped for the rapid estimation of microbial diversity and community compos ition in freshwater environments, Following isolation of total community DN A, PCR amplification of the 16S-23S intergenic spacer region in the rRNA op eron was performed with a fluorescence-labeled forward primer. ARISA-PCR fr agments ranging in size from 400 to 1,200 bp were next discriminated and me asured by using an automated electrophoresis system. Database information o n the 16S-23S intergenic spacer was also examined, to understand the potent ial biases in diversity estimates provided by ARISA. In the analysis of thr ee natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities wer e quantitatively compared in terms of both their relative diversity and sim ilarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41), In addit ion, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 3 0 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest t hat ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.