BIAS IN TEMPLATE-TO-PRODUCT RATIOS IN MULTITEMPLATE PCR

Bias introduced by the simultaneous amplification of specific genes fr om complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S r ibosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly r elated bacterial species were mixed and amplified with universal, dege nerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplifi cation of specific templates. Variability between replicates also cont ributed to the observed bias but in a comparatively minor way, Based o n these initial observations, template dosage and differences in bindi ng energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16 S 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 hig her efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification, In contrast, g ene 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 speci es were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing few er cycles, and by mixing replicate reaction preparations.