Sun, 27 Jul 2008 09:18:57 BST CiteULike: vrich's bias http://www.citeulike.org/user/vrich/tag/bias CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/vrich/article/2783559 <i>Appl. Environ. Microbiol., Vol. 64, No. 11. (1 November 1998), pp. 4522-4529.</i><br /><br />Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5' domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values. Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity Marcelino Suzuki Michael Rappe Stephen Giovannoni Appl. Environ. Microbiol., Vol. 64, No. 11. (1 November 1998), pp. 4522-4529. 2008-05-11T01:05:46-00:00 1998 Appl. Environ. Microbiol. 64 11 4522 4529 bias lh-pcr http://www.citeulike.org/user/vrich/article/2754508 <i>Nucl. Acids Res., Vol. 30, No. 9. (1 May 2002), pp. 2083-2088.</i><br /><br />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 Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR' Janelle Thompson Luisa Marcelino Martin Polz doi:10.1093/nar/30.9.2083 Nucl. Acids Res., Vol. 30, No. 9. (1 May 2002), pp. 2083-2088. 2008-05-04T21:02:22-00:00 2002 Nucl. Acids Res. 30 9 2083 2088 bias pcr