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Applied and Environmental Microbiology, October 1999, p. 4630-4636, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Automated Approach for Ribosomal Intergenic Spacer
Analysis of Microbial Diversity and Its Application to Freshwater
Bacterial Communities
Madeline M.
Fisher and
Eric W.
Triplett*
Department of Agronomy, Brock Institute for
Environmental Microbiology, University of Wisconsin
Madison, Madison,
Wisconsin 53706
Received 26 April 1999/Accepted 14 July 1999
An automated method of ribosomal intergenic spacer analysis (ARISA)
was developed for the rapid estimation of microbial diversity and
community composition in freshwater environments. Following isolation
of total community DNA, PCR amplification of the 16S-23S intergenic
spacer region in the rRNA operon was performed with a
fluorescence-labeled forward primer. ARISA-PCR fragments ranging in
size from 400 to 1,200 bp were next discriminated and measured by using
an automated electrophoresis system. Database information on the
16S-23S intergenic spacer was also examined, to understand the
potential biases in diversity estimates provided by ARISA. In the
analysis of three 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 were quantitatively compared in terms of both
their relative diversity and similarity level. The three communities
had distinctly different profiles but were similar in their total
number of fragments (range, 34 to 41). In addition, the pattern of
major amplification products in representative profiles was not
significantly altered when the PCR cycle number was reduced from 30 to
15, but the number of minor products (near the limit of detection) was
sensitive to changes in cycling parameters. Overall, the results
suggest that 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.
*
Corresponding author. Mailing address: Department of
Agronomy, University of Wisconsin
Madison, 1575 Linden Dr., Madison, WI 53706. Phone: (608) 262-9824. Fax: (608) 262-5217. E-mail: triplett{at}facstaff.wisc.edu.
Applied and Environmental Microbiology, October 1999, p. 4630-4636, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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