CiteULike: vrich pcr_bias

Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR

Suzuki — 2008-05-11T01:25:37-00:00

Applied and Environmental Microbiology, Vol. 62, No. 2., 625.

PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample.

SG Acinas — 2006-03-17T19:55:57-00:00

Appl Environ Microbiol, Vol. 71, No. 12. (December 2005), pp. 8966-8969.

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.

Uncultured archaea in deep marine subsurface sediments: have we caught them all?

Andreas Teske — 2008-04-18T19:56:37-00:00

ISME J, Vol. 2, No. 1. (November 2007), pp. 3-18.

Bias in Template-to-Product Ratios in Multitemplate PCR

Martin Polz — 2008-05-04T21:02:13-00:00

Appl. Environ. Microbiol., Vol. 64, No. 10. (1 October 1998), pp. 3724-3730.

Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.