Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA) |
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Authors: | Robin E Everts Serge A Versteeg Corinne Renier Francoise Vignaux Peter C Groot Jan Rothuizen Bernard A van Oost |
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Institution: | (1) Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Universiteit Utrecht, PO Box 80.154, 3508 TD, Utrecht, The Netherlands, NL;(2) UPR 41 CNRS Recombinaisons Génétiques, Faculté de Médecine, 2 avenue du Professeur Léon Bernard, 35043 Rennes Cedex, France, FR;(3) Department of Equine Sciences and Department of Farm Animal Health, Universiteit Utrecht, The Netherlands, NL |
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Abstract: | Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related
genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified
size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in
order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization,
a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after
the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the
third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that
approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round
difference product. About half of the clones identified in the second-round difference product were also present in the third-round
difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were
tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester
specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization
of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped
to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track
disease genes within lines of pedigree dogs.
Received: 26 April 2000 / Accepted: 11 May 2000 |
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