Comparison of DNA isolation methods from mammalian sperm cells and development of a new protocol

Abstract

Sperm DNA tightly packed with protamines makes the DNA isolation procedure from sperm cells long and laborious. Cell lysis is also a challenging step because of the disulfide bonds-rich membranes of the sperm cells. In this study we aimed to evaluate potential rapid DNA isolation protocols to isolate DNA from mammalian sperm cells, and develop an easy, rapid, and cost-effective protocol for sperm DNA isolation which can be used in molecular biology and diagnostics. Sperm samples were collected from seven adult rats. Our developed protocol included Proteinase K and small amount of β-mercaptoethanol (βME) for cell lysis. A modified salting-out technique was then employed to collect DNA. Alternative protocols involving InstaGene matrix and cell sonication techniques were also applied to achieve DNA isolation. Concentration of the DNA yield was measured, and the degradation of DNA was checked using agarose gel electrophoresis. The intactness of the DNA yield was assessed and validated using polymerase chain reaction (PCR) and capillary gel electrophoresis techniques. The lysis of the cells and high-quality DNA yield have only been achieved using our developed optimized protocol. To confirm the quality of DNA for assays, PCR product was synthesized for rat actin β (RActβ) gene and then analyzed using capillary gel electrophoresis. A strong peak at right m/z value for the amplicon was obtained. We described an improved protocol over the previous methods suggesting the use of combined commercial kits and long incubation times.