DNA extraction from tissues and subsequent PCR is something most biology labs do on a routine basis. First, there is the tissue collection and DNA prep and then the setup of the PCR for gene amplification, followed by analysis using gel electrophoresis. Usually the time-limiting step is the DNA extraction from the tissue itself. Standard protocols call for DNA lysis at 55°C in various buffers containing Proteinase K anywhere from 1-8 hours, with longer times or overnight incubations being the standard method. However, there are times when results are needed fast and it is not convenient to wait for lysis to occur. Sigma has come out with a kit that promises tissue prep in just 15 minutes!! This is followed by PCR setup that only requires the addition of water, primers and DNA and, as a bonus, can even be assembled at room temperature. This is definitely the fastest prep I have found.
The kit is optimized for use with mouse tails and other animal tissue, buccal swabs, hair shafts and saliva. I have only ever used it with mouse tails and tissue, so I can’t vouch for results from other sample types. After tissue collection, the DNA is extracted by addition of 100 µl Extraction Solution and 25 µl Tissue Preparation Solution (which can be pre-mixed before use for multiple samples). This is incubated at room temperature for 10 minutes and then incubated at 95°C for 3 minutes. 100 µl of a Neutralization Solution is then added and the sample is vortexed and ready for immediate PCR or storage at 4°C. The kit supplies a 2X PCR Reaction Mix that can be stored at 4°C, eliminating the wait for thawing of reagents. This Mix contains the buffer, salts, dNTPs and Taq Polymerase. The PCR can be assembled at room temperature with the addition of forward and reverse primer, water and the tissue extract. It can then be placed in the thermal cycler for amplification with standard cycling parameters.
Being responsible for a lot of mouse genotyping, I have used this kit several times with mouse tissue and tails. I have not had 100% success but it is pretty reliable. I do avoid using it with certain PCR protocols that are notoriously tricky, as the reaction doesn’t always work and it is always better to stay with something tried and true than to have to repeat the PCR. The one major drawback with this kit is that the DNA prep is supposed to be used only with Sigma’s RedTaq™ polymerase, which becomes the limiting step as the polymerase provided with the kit is used up long before the DNA extraction solutions (RedTaq™ polymerase can be obtained separately from Sigma, though). It becomes a pain to make sure these samples are separated from those which undergo a different extraction process so that the right polymerase is used. I have used the DNA a couple of times with Promega’s Taq polymerase, however, and the PCR seemed to work fine 95% of the time. But other than those drawbacks, there is nothing that I have found that beats the speed and simplification of this kit. For routine PCR reactions and genotyping, this is the fastest and most efficient method out there.
Research Associate
Department of Ophthalmology
University of California, San Francisco