A quantitative approach to detect and overcome PCR inhibition in ancient DNA extracts.
Inhibition is problematic in many applications of PCR, particularly those involving degraded or low amounts of template DNA, when simply diluting the extract is undesirable. Two basic approaches to monitoring inhibition in such samples using real-time or quantitative PCR (qPCR) have been proposed. The first method analyzes the quantification cycle (Cq) deviation of a spiked internal positive control. The second method considers variations in reaction efficiency based on the slopes of individual amplification plots. In combining these methods, we observed increased Cq values together with reduced amplification efficiencies in some samples, as expected; however, deviations from this pattern in other samples support the use of both measurements. Repeat inhibition testing enables optimization of PCR facilitator combinations and sample dilution such that DNA yields and/or quantitative accuracy can be maximized in subsequent PCR runs. Although some trends were apparent within sample types, differences in inhibition levels, optimal reactions conditions, and expected recovery of DNA under these conditions suggest that all samples be routinely tested with this approach.
King
Christine
C
McMaster Ancient DNA Centre, Department of Anthropology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada.
Debruyne
Régis
R
Kuch
Melanie
M
Schwarz
Carsten
C
Poinar
Hendrik
H
D000465:Algorithms; D003198:Computer Simulation; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004247:DNA