Trends in Microbiome and Digital One Health

When the polymerase chain reaction (PCR) is used to amplify simultaneously multiple templates, preferential amplification of certain templates (PCR bias) leads to a distorted representation of the original templates in the final amplicon pool. PCR selection, a type of PCR bias, is influenced by mismatches between primers and templates, the locations of mismatches, and the nucleotide pairing of mismatches. Direct measurement of primer-template interactions has not been possible, leading to uncertainty when attempting to optimize PCR reactions and degenerate primer pools. I will describe a novel method (‘Deconstructed PCR’) that has been used to generate empirical data about which primers anneal to which templates in complex systems and to reduce PCR bias by limiting the number of cycles of interaction between primers and genomic DNA templates. Deconstructed PCR is also shown to provide a mechanism for reducing chimeric PCR artifacts and for introducing absolute quantitation in historically compositional analyses. In controlled experiments with synthetic DNA templates, primer-template interactions are further explored to assess the effects of annealing temperature and primer combinations of perfect match and mismatch amplifications. These data are used to improve our ability to target a broader range of targets and reduce PCR bias when using domain-level primers for microbial community analysis.