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.
Highlights
Numerous impurities are inadvertently co-purified with DNA and can inhibit the polymerase chain reaction (PCR) [1,2,3]
PCR inhibitors can lead to inaccurate quantitative PCR results and, if present in sufficient quantities, these impurities can lead to PCR failure/false negatives
It is notable that PCR enhancer cocktail 1 (PEC-1) uniquely identified the 1124–1 full concentration sample as Chinook salmon and standard PCR uniquely identified the 1124–2 1:10 sample as Chinook salmon
Summary
Numerous impurities are inadvertently co-purified with DNA and can inhibit the polymerase chain reaction (PCR) [1,2,3]. Methods have been developed for the removal of PCR inhibitors during DNA extraction and purification. Repeat silica extraction has been demonstrated useful in this regard [7, 9] This method relies on repeated rounds of silicabased extraction that must remove PCR inhibitors at a rate faster than its associated loss of DNA [10]. Other methods in this category include extraction with thiopropyl sepharose 6B resin, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulphate (SDS), isopropanol precipitation, and/or polyvinylpyrrolidone (PVP) [1, 11,12,13,14]
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