CiteULike: vrich's pcr

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.

Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'

Janelle Thompson — 2008-05-04T21:02:22-00:00

Nucl. Acids Res., Vol. 30, No. 9. (1 May 2002), pp. 2083-2088.

Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated. Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR. Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE). Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample. 10.1093/nar/30.9.2083

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.