Improved DNA profiles from aged horse feces using pressure cycling technology
2016
Deshpande, K. | Villarreal, M. | Mills, D. K.
Feces represent an easily available, non-invasive source of DNA. Often DNA extraction from feces is compounded with contamination, dietary inhibitors and primarily low quantity and poor quality of host DNA. In an effort to improve host DNA recovery from fecal samples, a new DNA extraction methods was developed. The objectives of this project was to use non-invasive sampling techniques of feces from domestic horses, provide a viable protocol for an improved DNA extraction, and genotype using six equine microsatellite on feces aged for 6 days. The technique used a modified Qiagen® DNA Stool Mini Kit protocol (Q) with pressure cycling technology (PCT). The DNA yield using PCT extraction was five fold greater as compared to the Q. The PCT mediated Qiagen (Q + PCT) technique yielded complete (six loci) equine DNA profiles of 100 % samples ≤2 days old, 90 % at 4 days and 60 % at 6 days post defecation. The results indicated that the Q + PCT increased DNA yield, and thereby increased the likelihood of obtaining an equine DNA profile from aged fecal samples. Using ML-RELATE analyses, the profiles obtained from the six equine loci could also be used for kinship analysis. Samples aged up to Day 6 as well as pasture samples with unknown days since defecation were successful in individualization of the contributors and could also be used to analyze kinship. The Q + PCT method should prove to be an extremely useful and reliable method for conservation or forensic cases where fecal matter may be the only sample available for analyses.
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