Rescue PCR: Reagent-rich PCR recipe improves amplification of degraded DNA extracts

Abstract

Minimizing the inadvertent co-extraction of polymerase chain reaction (PCR) inhibitors and/or subduing their influence are two of the most pervasive challenges in the study of ancient DNA (aDNA). Some commonly employed methods to circumvent inhibition include dilution of DNA extracts and/or removal of inhibitors via silica-based treatments. While these methods have been shown to be effective, they may not be useful for all aDNA extracts. Samples with very low copy number, for instance, may not benefit from such methods, as dilutions lower DNA concentration in tandem with the inhibitors, and some DNA loss is expected to follow silica-based treatments. Therefore, the development of additional options to overcome PCR inhibition is at a premium. In this study, we present evidence that a reagent-rich PCR protocol, where all reagents are increased in equal relative proportion can increase amplification success when DNA concentration is reduced relative to inhibitors. The reagent-rich PCR recipe, termed rescue PCR, increased amplification success by 51% for the 112 extracts used in the study. Rescue PCR represents a simple and robust addition to the suite of options currently available to work with DNA in the presence of inhibition, especially ancient, degraded, and low copy number DNA extracts.