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Applied and Environmental Microbiology
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Microbial Ecology

PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities

Sven Becker, Peter Böger, Ralfh Oehlmann, Anneliese Ernst
Sven Becker
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Constance, and
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Peter Böger
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Constance, and
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Ralfh Oehlmann
PE Biosystems, Weiterstadt, and
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Anneliese Ernst
NIOO-Centre for Estuarine and Coastal Ecology, Yerseke, The Netherlands
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DOI: 10.1128/AEM.66.11.4945-4953.2000
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ABSTRACT

Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69–75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 104-fold excess of phylogenetically similar DNA and of an up to 107-fold excess of dissimilar DNA.

  • Copyright © 2000 American Society for Microbiology
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PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities
Sven Becker, Peter Böger, Ralfh Oehlmann, Anneliese Ernst
Applied and Environmental Microbiology Nov 2000, 66 (11) 4945-4953; DOI: 10.1128/AEM.66.11.4945-4953.2000

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PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities
Sven Becker, Peter Böger, Ralfh Oehlmann, Anneliese Ernst
Applied and Environmental Microbiology Nov 2000, 66 (11) 4945-4953; DOI: 10.1128/AEM.66.11.4945-4953.2000
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KEYWORDS

bacteria
Ecosystem
Polymerase Chain Reaction
Taq Polymerase

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