Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5 '-nuclease assays

Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has re lied mainly on oligonucleotide hybridization to extracted rRNA or intact rR NA in whole cells. However, low abundance and cellular rRNA content limit t he application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5'-nuclease assay, als o known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDN As) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and pr obe combinations for quantification of SSU rDNAs at the domain and group le vels were developed and tested for specificity and quantitative reliability . We examined the spatial and temporal variations of SSU rDNAs from Synecho coccus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5 '-nuclease assays reliably quantified rDNAs over at least 4 orders of magni tude and accurately measured the proportions of genes in artificial mixture s. The spatial and temporal distributions of planktonic microbial groups me asured by the 5'-nuclease assays were similar to the distributions estimate d by quantitative oligonucleotide probe hybridization, whole-cell hybridiza tion assays, and flow cytometry.