Optimizing genomic DNA isolation from chicken spermatozoa

R.R. Churchil, Anup Singh, Jaiyashi Gupta and Deepak Sharma
Division of Avian Genetics and Breeding Central Avian Research Institute
Izatnagar, Bareilly - 243 122

Genomic DNA can be isolated from all the tissues of nucleus containing cells. Although the basis of DNA purification is similar to all kinds of tissues, the extraction of sperm DNA require slight modifications of conventional DNA isolation methods since the nuclei of sperm cells are enclosed in a capsid heavily cross-linked by disulphide bridges. Chicken spermatozoa are greatly elongated and their volume is substantially reduced compared to bull’s sperm. In contrast to mammalian semen, chicken semen is less copious with scanty seminal plasma thereby highly concentrated and is easy to collect. For these reasons cock’s semen is a convenient source of genomic DNA. Moreover, DNA isolation from sperm is obligatory for some molecular techniques like sperm-mediated transgenesis. Keeping in view of the distinct characteristics of chicken semen, a simple and efficient protocol to isolate genomic DNA from chicken spermatozoa has been standardized. Briefly, neat semen containing 1.5 X 10 8 sperm cells was washed once with phosphate buffer saline (PBS) and pelleted by centrifugation. The sperm pellet was then resuspended in PBS by vortexing and prewarmed (55°C) sperm lysis buffer containing 2-mercaptoethanol was added. The mixture was then incubated at 55°C for 2 hrs. After the incubation, proteinase K (20mg/ml) was added and the incubation was continued overnight. The DNA was extracted using phenol: chloroform extraction methods and subsequently precipitated using chilled absolute ethanol, pelleted, washed in 70% alcohol and dissolved in TE buffer. On resolution in 0.8% agarose gel, single distinct band indicated the extraction of high quality DNA without shearing and RNA contamination. The DNA extracted by this method was suitable for PCR, southern hybridization and all other molecular applications.

Source : IPSACON-2005

Did You Like This Article? Bookmark it at Del.icio.us

Article Tools