Over the last 2 years my laboratory has been involved in a large-scale project examining the population genetics of wild felids. Over the last 20 years we have accumulated over 400 samples of species including bobcat, ocelot, and mountain lion. These samples were obtained during field studies, from road kills, and from museums, and therefore include everything from fresh blood stored in Longmire’s buffer to bone over 100 years old. My first challenge was to optimize a protocol that would be rapid, consistent and could be used with a wide range of starting material. The extracted DNA would be used as a template for PCR amplification of microsatellites and mtDNA regions.
I considered various DNA extraction methods and including spin-column based methods and phenol-chloroform based protocols, but was not satisfied with either of these. I needed a protocol that 1) did not use toxic compounds, 2) was fast and reliable with different starting materials, and 3) the reagents could be scaled down to extract maximum amount of samples.
I was referred to the Gentra Systems PUREGENE DNA Purification Kit for purifying genomic, mitochondrial, and viral DNA. The PUREGENE system works via alcohol and salt precipitation. The first step is to lyse cells with an anionic detergent in the presence of a DNA stabilizer that inhibits DNase activity, after which RNA is digested. The proteins are digested and removed along with other contaminants by salt precipitation. The DNA is then alcohol precipitated and dissolved in a DNA stabilizer. This protocol requires a very limited number of centrifugation steps and there for is rapid, taking approximately 2 hours of actual working time per 24 samples. This kit provides very consistent results and produces high yields of DNA comparable to, or higher than, the two extraction methods I previously used.
I initially tested a trial kit and since then I have used this kit to successfully extract genomic and mitochondrial DNA (mtDNA) from liver, blood, tissue, hair, scat, and bone. Because this protocol is based entirely on solutions, I was also able down scale the amount of reagent I use so that I can extract maximum amount of samples per kit. I have used the extracted DNA in PCR amplification of 16 microsatellites and 1 mtDNA region with very consistent results. In addition, the customer service department is efficient and their technical department is very knowledgeable. Their technicians have helped me optimize my protocols to provide high yield from degraded samples such as hair, bone, and scat. This company also has great website where they post many protocols and reports on research that utilizes their kits. Finally, PUREGENE kit prices are comparable to other DNA extraction kits, but they can be used to extract more samples making them more economical.
I would recommend this product to anyone who wishes to extract large quantities of DNA from different types of tissue, and does not want to use toxic reagents. I give this company and product a 9 on a scale of 1 – 10. The only improvement would be to make this extraction compatible with vacuum type systems so 96 samples could be extracted at a time and the number of samples extracted would not be limited by space available in a centrifuge.
Jan E. Janecka
PhD Student
Feline Research Program
Caesar Kleberg Wildlife Research Institute
Texas A&M University-Kingsville