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Applied and Environmental Microbiology
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Methods

Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

Marcelino T. Suzuki, Lance T. Taylor, Edward F. DeLong
Marcelino T. Suzuki
Monterey Bay Aquarium Research Institute, Moss Landing, California 95039
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Lance T. Taylor
Monterey Bay Aquarium Research Institute, Moss Landing, California 95039
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Edward F. DeLong
Monterey Bay Aquarium Research Institute, Moss Landing, California 95039
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DOI: 10.1128/AEM.66.11.4605-4614.2000
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ABSTRACT

Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plusProchlorococcus 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 magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.

  • Copyright © 2000 American Society for Microbiology
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Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays
Marcelino T. Suzuki, Lance T. Taylor, Edward F. DeLong
Applied and Environmental Microbiology Nov 2000, 66 (11) 4605-4614; DOI: 10.1128/AEM.66.11.4605-4614.2000

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Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays
Marcelino T. Suzuki, Lance T. Taylor, Edward F. DeLong
Applied and Environmental Microbiology Nov 2000, 66 (11) 4605-4614; DOI: 10.1128/AEM.66.11.4605-4614.2000
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