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.

Sex Determination of Sika Deer (Cervus nippon yesoensis) Using Nested PCR from Feces Collected in the Field

“…Almost all the outer pellet layer of each fecal pellet scraped with a surgical blade was applied as fecal material. Since the weight of feces changes depending on its moisture content and, as has been suggested by Wehausen et al (2004), the outer pellet layer contains a significant amount of high quality template DNA. Second, 1.5 ml of Buffer ASL was added to each fecal sample to accumulate enough supernatant following centrifugation, while a pestle (LMS, Tokyo, Japan) was used to crush fecal material to obtain the maximum DNA concentration.…”

“…In this situation, fecal samples that can be collected noninvasively are simply counted instead of biomaterials and studies using fecal samples have been conducted in many species [2,5,11,23,25,30]. To use fecal samples for further analysis beyond genotyping, sex information will be essential.…”

The Genetic Diversity of Tibetan Red Deer Populations Determined using Mitochondrial and Microsatellite Markers

“…Samples with shallow surface colors possess relatively little pigment and the quality and quantity of DNA extracted are higher than from those samples with deep surface color. For Tibetan red deer, the optimal method for extracting DNA is to use the outermost layer of the fecal pellets, which seems to maximize DNA yield and minimize inhibitor content (Wehausen et al, 2004). The fecal DNA extraction process used the DNA Stool Mini Kit by QIAGEN (QIAGEN, Hilden, Germany), following the manufacturer's instructions.…”

Species identification, molecular sexing and genotyping using non‐invasive approaches in two wild bovids species: Bos gaurus and Bos javanicus

“…Using DNA extracted from non-pellet herbivore feces, the main difficulties included low-quantity DNA template or/and DNA degradation owing to degenerative environmental processes [Wehausen et al, 2004]. Tropical climatic conditions encountered in Vietnam such as high temperature, humidity and a high UV radiation must contribute strongly to the degradation of cellular DNA.…”

“…Moreover, in tropical environmental conditions the use of such methods is particularly problematic as cells are much more exposed to degenerative processes [Wehausen et al, 2004]. In the case of herbivores, the use of fecal DNA poses further problems owing to the inhibitory effects that some plant secondary compounds exert on PCR polymerases.…”