Subduing the influence of PCR inhibitors on amplifying aged, degraded, and low copy number DNA: PCR enhancer cocktail-p and rescue PCR

Abstract

PCR inhibitors are a formidable problem to the study of aged, degraded, and/or low copy number DNA. As a result, there is a need to find alternate methods that ameliorate the efficacy of PCR. In this study, we attempted to use genetic methods to identify the species of salmonid (Oncorhynchus spp.) remains recovered from archaeological sites along the Feather River located in northern California, United States. In the process of doing so, we compared the efficacy of a PCR enhancer cocktail called "PEC-P" and a reagent rich PCR recipe called "rescue PCR" over standard PCR. Across all treatments (full concentration and 1:10 dilute eluates subjected to standard PCR, PEC-P, and rescue PCR) species identification was possible for 74 of 93 archaeological fish specimens (79.6%). Overall, six of the 93 samples (6.5%) consistently yielded species identification across all treatments. The species of ten specimens (10.8%) were uniquely identified from amplicons produced with either PEC-P or rescue PCR or both. Notably, the species of seven samples (7.5%) were uniquely identified with standard PCR over the alternative treatments. Considering both full concentration and 1:10 dilute eluates (N = 186), standard PCR performed as well as PEC-P (p = 0.1451) and rescue (p = 0.6753). Yet, considering results from full concentration eluates alone (N = 93), PEC-P (60.2%) outperformed both standard PCR (44.1%; p = 0.0277) and rescue PCR (40.9%; p = 0.0046). Stochasticity observed in our study cautions us against choosing a "best" performing method of those explored here and suggests their respective potentials to improve success may be sample dependent. When working with samples compromised by PCR inhibitors, it is useful to have alternative methodologies for subduing the problem. Both PEC-P and rescue PCR represent useful alternative methods for the study of aged, degraded, and/or low copy number DNA samples compromised by PCR inhibitors.

Evaluating genotyping‐in‐thousands by sequencing as a genetic monitoring tool for a climate sentinel mammal using non‐invasive and archival samples

“…Though preliminary quality checks demonstrated the presence of whole genomic DNA extracted from fecal pellets, it is likely that the concentration of target species DNA in these samples was very low. As PCR amplification is a stochastic process, samples with very low starting concentrations of target DNA and/or that contain PCR inhibitors may differentially amplify alleles (Kebschull & Zador, 2015 ; Kemp et al., 2020 ), leading to high variance of allele read counts. The broader distribution of allele count ratios within fecal pellet samples compared to tissue samples indicated that differential allele amplification was likely the driving factor of the high genotyping error observed between replicates (Figure 4 ).…”

“…hair and feces) and archival (e.g. skins, skeletons, or fluid‐preserved specimens in natural history museum collections) samples that have historically presented challenges for sequencing success and genotyping accuracy due to its low quantity/quality (Andrews et al., 2021 ; Raxworthy & Smith, 2021 ) and presence of PCR inhibitors (Kemp et al., 2020 ; Monteiro et al., 1997 ). Additionally, there is often an abundance of exogenous DNA within non‐invasive and archival samples, especially from microbial sources, which may cause off‐target co‐amplification during PCR that obscures the targeted signal (Carpenter et al., 2013 ; Perry et al., 2010 ; Taberlet et al., 1999 ).…”

Freshwater and anadromous fishing in Ice Age Beringia

“…Furthermore, we also used a PCR enhancer cocktail called PEC-P (DNA Polymerase Technology) in additional PCRs. This enhancer has been found to be useful in the amplification of aDNA ( 90 , 93 ). Fifteen microliters PEC-P PCR reactions contained 1× Omni Klentaq reaction buffer, 0.32 mM dNTPs, 0.24 μM each primer, 0.3 U of Omni Klentaq LA polymerase, 20% (v/v) PEC-P, and 1.5 μl of template DNA.…”

Ancient DNA Identification of Giant Snakehead (Channa micropeltes) Remains from the Market Street Chinatown and Some Implications for the Nineteenth-Century Pacific World Fish Trade

“…First, “standard” PCRs contained 1X Omni Klentaq Reaction Buffer, 0.32 mM dNTPs, 0.24 μM of each primer, 0.3 U of Omni Klentaq LA polymerase, and 1.5 μL of template DNA. Second, we employed a PCR buffer cocktail called PEC-P (DNA Polymerase Technology) that has been found useful in amplifying aged and degraded DNA (Kemp et al 2020; Palmer et al 2018). PEC-P PCR reactions with Klentaq were 15 μL each, containing 1X Omni Klentaq Reaction Buffer, 0.32 mM dNTPs, 0.24 μM of each primer, 0.3 U of Omni Klentaq LA polymerase, 20% (v/v) PEC-P, and 1.5 μL of template DNA.…”