Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis
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Open Access
- 1 May 2007
- journal article
- Published by Oxford University Press (OUP) in FEMS Microbiology Ecology
- Vol. 60 (2), 341-350
- https://doi.org/10.1111/j.1574-6941.2007.00283.x
Abstract
In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using ‘universal’ bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61°C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.Keywords
This publication has 30 references indexed in Scilit:
- Comparison of RNA- and DNA-based species diversity investigations in rhizoplane bacteriology with respect to chloroplast sequence exclusionJournal of Microbiological Methods, 2004
- Review and re-analysis of domain-specific 16S primersJournal of Microbiological Methods, 2003
- Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environmentFEMS Microbiology Letters, 2003
- An evaluation of terminal‐restriction fragment length polymorphism (T‐RFLP) analysis for the study of microbial community structure and dynamicsEnvironmental Microbiology, 2000
- Genome economization and a new approach to the species concept in bacteriaProceedings. Biological sciences, 1999
- Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecologyAntonie van Leeuwenhoek, 1998
- Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone librariesMolecular Ecology, 1997
- Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions.Genome Research, 1993
- Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populationsApplied and Environmental Microbiology, 1990
- Alteration of the specificity of PvuII restriction endonucleaseNucleic Acids Research, 1987