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Applied and Environmental Microbiology, November 2000, p. 4945-4953, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Sven Becker,1,* Peter Böger,1 Ralfh Oehlmann,2 and Anneliese Ernst3

Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Constance,1 and PE Biosystems, Weiterstadt,2 and NIOO-Centre for Estuarine and Coastal Ecology, Yerseke, The Netherlands3

Received 14 April 2000/Accepted 3 September 2000

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.

* Corresponding author. Mailing address: Universität Konstanz, Lehrstuhl für Physiologie und Biochemie der Pflanzen, 78457 Constance, Germany. Phone: 49-7531-883669. Fax: 49-7531-883042. E-mail: Sven.Becker{at}uni-konstanz.de.

dagger Publication 2667 of the NIOO-Centre for Estuarine and Coastal Ecology.

Applied and Environmental Microbiology, November 2000, p. 4945-4953, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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