Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method.

Abstract

Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition.