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dc.contributor.authorPolz, Martin F.
dc.contributor.authorCavanaugh, Colleen Marie
dc.date.accessioned2015-04-03T21:04:28Z
dc.date.issued1998
dc.identifier.citationPolz, Martin F., and Colleen M. Cavanaugh. 1998. "Bias in Template-to-Product Ratios in Multitemplate PCR." Applied and Environmental Microbiology 64, no. 10: 3724-3730.en_US
dc.identifier.issn0099-2240en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:14368998
dc.description.abstractBias 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.en_US
dc.description.sponsorshipOrganismic and Evolutionary Biologyen_US
dc.language.isoen_USen_US
dc.publisherAmerican Society for Microbiologyen_US
dc.relation.isversionofhttp://aem.asm.org/content/64/10/3724.full.pdf+htmlen_US
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC106531/en_US
dash.licenseLAA
dc.titleBias in Template-to-Product Ratios in Multitemplate PCRen_US
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden_US
dc.relation.journalApplied and Environmental Microbiologyen_US
dash.depositing.authorCavanaugh, Colleen Marie
dc.date.available2015-04-03T21:04:28Z
dash.contributor.affiliatedCavanaugh, Colleen


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