AME 26:13-22 (2001)  -  doi:10.3354/ame026013

ABSTRACT: Collection of microbial biomass and extraction of DNA are the first steps of many molecular approaches for examining uncultured microbes in aquatic ecosystems. Because of the difficulties of using large samples (up to 20 l) and the occasional ineffectiveness of DNA isolation procedures, we examined an alternative approach, Œfilter PCR¹, which consists of filtering small volumes through polycarbonate filters and using sections of a filter directly in PCR. Positive amplification was achieved with as little as 25 μl of coastal seawater, corresponding to about 10000 bacterial cells, although larger volumes (1 to 10 ml, depending on bacterial abundance) gave more consistent results. Denaturing gradient gel electrophoresis (DGGE) revealed few differences in the 16S rRNA amplicons from filter PCR and from the standard approach using DNA isolated from several liters of coastal seawater. A clone library of 16S rRNA amplicons from filter PCR was slightly more diverse than a clone library constructed by the standard approach. These results allow us to explore variation in microbial community structure over a range of spatial scales and to examine the relative evenness of microbial communities in aquatic habitats. Our results indicate that filter PCR is as effective as the standard approach in retrieving bacterial genes from uncultured microbes in aquatic environments.

KEY WORDS: Bacterial community structure · Filter PCR · DGGE · Clone libraries · 16S rRNA