Evaluating PCR Bias Through Experimental Investigations of Complex Primer-Template Interactions
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The polymerase chain reaction (PCR) is a well-established tool for amplification of regions of DNA and is used in a broad range of biological studies. PCR bias, in which some templates within a mixture of templates are preferentially amplified, is a well-known phenomenon. Despite substantial effort invested into correcting such bias, PCR-based studies continue to generate data that distort underlying template ratios. A major source of PCR bias is from primer-template interactions, leading to PCR selection favoring certain templates. Motives of this study were to understand better the causes of selection bias in PCRs with complex templates and complex degenerate primer pools, and to develop novel strategies to decrease bias. An experimental system was developed to reduce PCR bias by separating linear copying of templates from exponential amplification of amplicons (Deconstructed PCR or ‘DePCR’), and this system also provides a mechanism to quantify primer-template interactions (Primer utilization profiles or ‘PUPs’). DePCR was used to interrogate mock DNA communities and complex environmental samples, and all reactions were compared to standard PCR workflows. Experiments with annealing temperature gradients demonstrated a strong negative correlation between annealing temperature and the evenness of primer utilization in complex pools of degenerate primers. Critically, shifting primer utilization patterns mirrored shifts in observed microbial community structure. In experiments with mock DNA templates, DePCR demonstrates that although perfect match primer-template interactions are abundant, the dominant type of primer-template interactions are mismatch interactions, and mismatch amplification starts immediately during the first cycle of PCR. Furthermore, in DePCR reactions involving multiple mismatches, no strong effect on template profiles was observed. DePCR allows improved representation of templates, greater tolerance for mismatches between primers and templates, and greater success in amplifying complex templates with low complexity primer pools. In addition, PUPs are empirical quantitative data derived from primer interactions with genomic DNA templates, and are a novel form of biological information that can be acquired only with DePCR. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, has applicability to amplicon-based microbiome studies, and may also serve in other PCR-based protocols where primers and templates have mismatches.