KNAW Repository

PCR bias in ecological analysis: A case study for quantitative Taq nuclease assays in analyses of microbial communities

Becker, S. and Boger, P. and Oehlmann, R. and Ernst, A. (2000) PCR bias in ecological analysis: A case study for quantitative Taq nuclease assays in analyses of microbial communities. Applied and Environmental Microbiology, 66, 4945-4953. ISSN 0099-2240.

[img]PDF - Published Version
Restricted to KNAW only

1214Kb

Official URL: http://dx.doi.org/10.1128/AEM.66.11.4945-4953.2000

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 Tag 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 10(4)- fold excess of phylogenetically similar DNA and of an up to 10(7)-fold excess of dissimilar DNA. [KEYWORDS: Polymerase-chain-reaction; aquaticus dna-polymerase; ribosomal- rna genes; real-time pcr; planktothrix rubescens; fluorogenic probes; lake constance; messenger-rna; amplification; diversity]

Item Type:Article
Institutes:Nederlands Instituut voor Ecologie (NIOO)
ID Code:10834
Deposited On:24 Nov 2011 01:00
Last Modified:15 Jan 2014 09:33

Repository Staff Only: item control page