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

Succession of ecotypes, physiologically diverse strains with negligible rRN A sequence divergence, may explain the dominance of small, red-pigmented (p hycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep la kes (C, Postius and A. Ernst, Arch. Microbiol, 172:69-75, 1999), In order t o test this hypothesis, it is necessary to determine the abundance of speci fic ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Tag nuclease assa ys (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 sam ples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleot ides and due to accumulation of amplicons that are not detected by the TaqM an probe. A decrease in reaction efficiency, which can be recognized by dir ect monitoring of amplification, provides experimental evidence for the pre sence of such a problem and emphasizes the need for real-time technology in quantitative PCR, Use of specific primers and probes and control of amplif ication efficiency allow correct quantification of target DNA in the presen ce of an up to 10(4)-fold excess of phylogenetically similar DNA and of an up to 10(7)-fold excess of dissimilar DNA.