SPECIFIC CLONING OF DNA FRAGMENTS UNIQUE TO THE DOG Y-CHROMOSOME

A novel technique that enabled the specific cloning of a DNA fragment unique to the dog Y chromosome is described. The method involves compe titive hybridization of DNA prepared from male dog lymphocytes with bi otin-labeled DNA prepared from female dog lymphocytes. The biotinylate d female-female and male-female hybrid DNA fragments were removed by c apture with streptavidin-coated paramagnetic particles. Full-length do uble-stranded DNA was generated from the remaining fragments by using the Klenow fragment of DNA polymerase I, followed by direct cloning us ing a low-background ligation technique. Analysis of putative recombin ant clones derived by this method has led to the identification of a f ragment that hybridizes specifically to male dog DNA. The clones were selected initially on the basis of a differential signal obtained when hybridized to dilutions of male and female dog DNA immobilized on neu tral nylon membrane. To evaluate its suitability as a probe for trans- sexually grafted cells in transplantation studies, the fragment was la beled with digoxigenin and hybridized in situ to male and female dog t issue sections. The clone designated number 6.2 hybridized strongly to male dog nuclei. The cloning strategy employed could be extended to o ther studies in which competitive reassociation can be used to identif y unique DNA sequences.