Bias in Template-to-Product Ratios in Multitemplate PCR

Abstract

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.

Variation in Dissolved Organic Matter Controls Bacterial Production and Community Composition

“…PCR a m p l i fi c a t i o n u s e d p r i m e r 3 5 7 f ( gþc ) ( 5 0 -CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGC-CCCCGCCCCCCTACGGGAGGCAGCAG-3 0 ), which contains a GC clamp and is specific for most b a c t e r i a , a n d u n i v e r s a l p r i m e r 5 1 9 r ( 5 0 -ACCGCGGCTG CTGGCAC-3 0 ). We minimized the problem of under-or over-amplification of particular gene sequences (Suzuki and Giovanoni 1996) by carefully controlling the reaction parameters (Polz and Cavanaugh 1998). Table 1) in the order ''inoculum/DOM.''…”

Novel hopanoid cyclases from the environment

“…Amplification by PCR can cause proportional biases during amplification (e.g. Polz and Cavanaugh, 1998). The primers used here also did not target Planctomycetes; nor would any of the primer combinations capture taxa similar to sequence 9SS, which contains the AA motif SPVWDS in the forward primer location (rather than SPVWDT).…”

Assessing the utility of metabarcoding for diet analyses of the omnivorous wild pig (Sus scrofa)

“…The only caveat in using CO1 for diet analysis within wild pigs, or any vertebrate host, is the co-amplification of host DNA along with diet. Host DNA template is more abundant and less degraded than DNA from diet items (Deagle, Eveson, & Jarman, 2006;Nejstgaard et al, 2008;Vestheim & Jarman, 2008), which can bias or restrict the molecular detection of food items (Green & Minz, 2005;Polz & Cavanaugh, 1998). Given these challenges, we also investigated the utility of blocking primers to limit the co-amplification and sequencing of the host CO1 DNA (Vestheim & Jarman, 2008).…”