MODIFIED GUANIDINIUM THIOCYANATE METHOD FOR HUMAN SPERM DNA ISOLATION

Mammalian sperm chromatin is highly condensed, so isolating DNA from s uch chromatin can be a formidable task. The procedures that produce hi gh quality DNA from somatic cells fail to yield quality sperm DNA. In this study we have modified the previously used guanidinium method to make it simple and efficient in isolating human sperm DNA. In our meth od, the lysis buffer contained guanidinium, sodium citrate, sarkosyl, proteinase K and mercaptoethanol. Proteinase K was not used in the ori ginal guanidinium method but was included in our protocol. CsCl centri fugation of the lysate, as described in the original procedure, was om itted. Instead, isopropyl alcohol was added directly to the lysis buff er to harvest the DNA. This modified guanidinium method generated high molecular weight DNA while the other two methods resulted in consider able DNA degradation. There was no difficulty in restriction enzyme di gestion of DNA prepared by the modified method as revealed by Southern blot analysis. Since the modified guanidinium method is a simple one- step procedure which avoids homogenization, organic solvents, centrifu gation and, more importantly, produces degradation-free DNA, it could be the method of choice when DNA from mature germ cells is needed.