A quantitative approach to detect and overcome PCR inhibition in ancient DNA extracts

Authors: C. King, R. Debruyne, M. Kuch, C. Schwarz, H. Poinar
Journal: BioTechniques, doi:10.2144/000113244

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.

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Time-Dependency of Molecular Rates in Ancient DNA Datasets, a Sampling Artifact?

Authors: R. Debruyne, H. N. Poinar
Journal: Systematic Biology, doi:10.1093/sysbio/syp028

It is common knowledge that the instantaneous rate of mutation (RoM) in DNA sequences exceeds the long-term rate of substitution (RoS) when measured in interspecific phylogenetic analyses. The neutral theory of molecular evolution describes this temporary excess diversity as transient polymorphisms either removed from the population through the actions of purifying selection or fixed by random genetic drift over a few generations (Kimura 1983). Observations of these “accelerations” in the molecular rates within recent evolutionary time have been documented (Parsons et al. 1997; Lambert et al. 2002); however, they did not resolve the magnitude and duration of this phenomenon. Howell et al. (2003) have addressed these issues through pedigree analyses of human mitochondrial (mt) hypervariable region (HVR) sequences and have suggested a 5- to 10-fold acceleration compared with the long-term RoS. In addition, Burridge et al. (2008) have shown that the calibration of the mt clock for galaxiid fishes using geological divergence dates with cytochrome b and control region sequences supports a transition period during which the RoM would decrease toward the RoS extending up to ~200 kyr (Burridge et al. 2008). However, the general applicability of these specific results remains untested…

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