Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons
Egert, M.G.G. ; Friedrich, M.W. - \ 2005
Journal of Microbiological Methods 61 (2005)1. - ISSN 0167-7012 - p. 69 - 75.
microbial community structure - length-polymorphism analysis - ribosomal-rna - genes - heteroduplexes - artifacts - mixtures - cloning
Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) analysis can be biased by the occurrence of pseudo-T-RFs, i.e., restriction fragments occurring in addition to the expected terminal restriction fragments of single amplicons. This bias originates from PCR products, which are single-stranded at their terminal restriction site. Here we show that treatment of PCR amplicons with Klenow fragment prior to restriction digest and T-RFLP analysis minimized effectively the occurrence of pseudo-T-RFs. Klenow fragment activity filled in bases into the partially single-stranded amplicons and thereby restored the affected amplicons to complete double strands. Our method allowed to improve the assessment of genetic diversity and gene ratios from T-RFLP analysis of an original environmental sample. Since partially single-stranded amplicons might influence many PCR-based techniques, post-amplification treatment with Klenow fragment may be useful for a wide range of applications, which assess the composition of amplicon pools, e.g., the analysis of marker gene diversity in mixed template samples by fingerprinting techniques or the analysis of sequence diversity by cloning.
Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences
Speksnijder, A.G.C.L. ; Kowalchuk, G.A. ; Jong, S. de; Kline, E. ; Stephen, J.R. ; Laanbroek, H.J. - \ 2001
Applied and Environmental Microbiology 67 (2001)1. - ISSN 0099-2240 - p. 469 - 472.
ribosomal-rna genes - polymerase chain-reaction - chimeric molecules - dna amplification - escherichia-coli - heteroduplexes - populations - coamplification - consequence - diversity
A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries