Dissecting primer editing by proofreading polymerases

Proofreading polymerases have 3 prime to 5 prime exonuclease activity that allows the excision and correction of misincorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards reveal the extent of editing and allow the comparison of different polymerases and cycling conditions. We further examine primer editing in the context of microbiome samples.