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Specific cloning of DNA fragments unique to the dog Y chromosome

Fletcher, S., Darragh, D., Fan, Y., Grounds, M.D., Fisher, C.J. and Beilharz, M.W. (1993) Specific cloning of DNA fragments unique to the dog Y chromosome. Genetic Analysis: Biomolecular Engineering, 10 (3-4). pp. 77-83.

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A novel technique that enabled the specific cloning of a DNA fragment unique to the dog Y chromosome is described. The method involves competitive hybridization of DNA prepared from male dog lymphocytes with biotin-labeled DNA prepared from female dog lymphocytes. The biotinylated female-female and male-female hybrid DNA fragments were removed by capture with streptavidin-coated paramagnetic particles. Full-length double-stranded DNA was generated from the remaining fragments by using the Klenow fragment of DNA polymerase I, followed by direct cloning using a low-background ligation technique. Analysis of putative recombinant clones derived by this method has led to the identification of a fragment 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 neutral nylon membrane. To evaluate its suitability as a probe for trans-sexually grafted cells in transplantation studies, the fragment was labeled with digoxigenin and hybridized in situ to male and female dog tissue sections. The clone designated number 6.2 hybridized strongly to male dog nuclei. The cloning strategy employed could be extended to other studies in which competitive reassociation can be used to identify unique DNA sequences.

Publication Type: Journal Article
Publisher: Elsevier B.V.
Copyright: © 1993 Published by Elsevier B.V.
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