Evaluation of methods that subdue the effects of polymerase chain reaction inhibitors in the study of ancient and degraded DNA

This study evaluated techniques that can potentially decrease time, cost, and labor in eliminating, circumventing, and/or inactivating as many polymerase chain reaction (PCR) inhibitors as possible while preserving DNA yield. In order to further explore the role of PCR inhibitors in ancient DNA (aDNA) studies, 140 DNA extractions were conducted on 112 Pacific salmonid vertebrae recovered from two archaeological sites located at Dionisio Point on Galiano Island, in southwestern British Columbia, Canada. These DNA extracts and their dilutions at 1:10 and 1:50 were screened for the presence of a 189 base pair (bp) portion of the 12S mitochondrial gene that is used for species identification of Pacific salmonids and other fish. These extracts and their dilutions were also screened for the presence of PCR inhibitors that can cause negative results for amplification of salmonid mtDNA. Repeat silica extraction was conducted on the full concentration extracts until: (1) they either produced a positive result in the salmonid mtDNA reaction, or (2) were deemed to be free of inhibition, but failed to amplify in the salmonid mtDNA reaction. In the latter case, the samples were concluded to not contain sufficient salmonid mtDNA to permit PCR amplification. In obtaining positive salmonid species identification, repeat silica purified extracts (81/133 successes) statistically outperformed dilutions at 1:10 (55/140, p=0.0018), 1:50 (63/140, p=0.0312), as well as across all dilutions combined (118/280, p=0.0025). We also explored the efficacy of EGTA as a decalcifying agent as compared to EDTA, which is commonly employed in aDNA studies. The only extracts that amplified at full concentration (6/140, 4.3%) were those in which EGTA decalcification was used. However, when diluted there was no statistical difference in the success of obtaining positive species identification from bone decalcified with EGTA or EDTA (1:10 and 1:50 dilutions, p=0.7891 and p> 0.9999 respectively). From the results of this study, we recommend that aDNA researchers employ greater flexibility in their methodologies as well as be cognizant of the role that PCR inhibitors may play in their investigations.