Differex™ System Technical Bulletin

Literature # TBD020

The Differex™ System uses the standard proteinase K digestion and a combination of phase separation and differential centrifugation for the separation of sperm and epithelial DNA. After a standard proteinase K digestion, the sample and buffer are placed in a spin basket seated in a tube containing a nonaqueous, nontoxic, biodegradable Separation Solution. This solution is denser than, and nonmiscible with, the aqueous buffer but is less dense than sperm. During centrifugation, the sperm pellets to the bottom of the tube. The aqueous solution containing the epithelial DNA remains on top of the nonaqueous solution. Removal of the aqueous solution is aided by the yellow color of the buffer. Only a thin film of the aqueous solution usually remains.This contaminating solution is easily removed by adding water to dilute the remaining DNA solution. The water is not miscible with the bottom solution and can be easily removed without additional centrifugation. The use of a dense nonaqueous fluid instead of a membrane eliminates clogging issues, and the nonaqueous nature of the solution helps to form a tight sperm pellet and keep cell debris away from the sperm pellet.

Following the efficient removal of the solution containing the epithelial DNA, the sperm DNA is isolated using the DNA IQ™ System(Cat.# DC6701). DNA IQ™™Lysis Buffer containing DTT is added to the pellet to dissolve the nonaqueous solution and lyse the sperm. The DNA IQ™™Resin is then added, and the sperm DNA is purified following the DNA IQ™ purification protocol. The epithelial DNA is purified in a similar manner by adding two volumes of DNA IQ™ Lysis Buffer with DTT and the DNA IQ™ Resin.

The total time for separating sperm from epithelial cells after addition of the sample to the proteinase K-containing Digestion Solution is approximately 90 minutes, which includes a 60-minute proteinase K digestion. DNA purification requires 30 minutes, so separation and purification can be accomplished in as little as 2 hours. Although more time is required, DNA can also be purified from the separated epithelial and sperm fractions using
phenol:chloroform-based methods.

The Differex™™System provides an easy and efficient method of removing the solution containing the epithelial DNA. Variability between examiners should be reduced. In addition, since only one centrifugation is required, fewer sperm should be lost during the process. Finally, the familiar proteinase K digestion from the standard methodology is retained for the upfront processing of the solid support.