Modified guanidinium thiocyanate method for human sperm DNA isolation.
Mammalian sperm chromatin is highly condensed, so isolating DNA from such chromatin can be a formidable task. The procedures that produce high 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 method, the lysis buffer contained guanidinium, sodium citrate, sarkosyl, proteinase K and mercaptoethanol. Proteinase K was not used in the original guanidinium method but was included in our protocol. CsCl centrifugation of the lysate, as described in the original procedure, was omitted. Instead, isopropyl alcohol was added directly to the lysis buffer to harvest the DNA. This modified guanidinium method generated high molecular weight DNA while the other two methods resulted in considerable DNA degradation. There was no difficulty in restriction enzyme digestion 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, centrifugation and, more importantly, produces degradation-free DNA, it could be the method of choice when DNA from mature germ cells is needed.
Hossain
A M
AM
Department of Obstetrics and Gynecology, University of South Alabama, Mobile 36688, USA.
Rizk
B
B
Behzadian
A
A
Thorneycroft
I H
IH
D004247:DNA / Q000302:isolation & purification*; D006146:Guanidines; D006801:Humans; D008297:Male; D008722:Methods; D013094:Spermatozoa; D013861:Thiocyanates
D006146:Guanidines; D013861:Thiocyanates; C054436:guanidine thiocyanate; D004247:DNA