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Journal of Heredity Advance Access originally published online on October 26, 2005
Journal of Heredity 2005 96(7):836-842; doi:10.1093/jhered/esi112
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© The American Genetic Association. 2005. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

High-Resolution Characterization of the Canine DLA-DRB1 Locus Using Reference Strand-Mediated Conformational Analysis

L. J. Kennedy, S. Quarmby, N. Fretwell, A. J. Martin, P. G. Jones, C. A. Jones, and W. E. R. Ollier

From the Centre for Integrated Genomic Medical Research, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, UK (Kennedy, Quarmby, Ollier); and the WALTHAM Centre for Pet Nutrition, Leicestershire, England (Fretwell, Martin, Jones, Jones)

Address correspondence to Lorna J. Kennedy at the address above, or e-mail: Lorna.Kennedy{at}manchester.ac.uk.

Several methods exist for genotyping class II DLA gene polymorphisms in the dog. The most accurate method is sequence-based typing, which involves direct sequencing of polymerase chain reaction products. However, this method is expensive and unsuitable for large-scale studies. Recently, reference strand-mediated conformation analysis (RSCA) has been shown to be effective for characterizing major histocompatibility complex genes in humans, sheep, horse, and cats. RSCA is a cheap and rapid method, ideal for large epidemiological studies. We have developed RSCA for typing DLA-DRB1 in the dog. Control panels including dogs typed by sequence-based typing and cloned major histocompatibility complex class II alleles in plasmids were used to establish migration patterns for each allele using 20 different fluorescent labeled references, of which 5 were selected to allow for clear identification and discrimination of all known DLA-DRB1 alleles. We have compared 168 dogs typed by RSCA for DLA-DRB1 and characterized by sequence-based typing, with less than 1% discrepancy. These differences were due to missing alleles because of a weak polymerase chain reaction. To date, we have RSCA-typed 1,394 dogs. RSCA is likely to become the method of choice for characterizing DLA genes in the dog and will prove a useful tool for dissecting the immune response of dogs in clinical studies.


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