title: High-resolution characterization of the canine DLA-DRB1 locus using reference strand-mediated conformational analysis.
abstract: 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.
authors.affiliation: Centre for Integrated Genomic Medical Research, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, UK. Lorna.Kennedy@manchester.ac.uk
authors.forename: L J
authors.initials: LJ
authors.lastname: Kennedy
authors.forename: S
authors.initials: S
authors.lastname: Quarmby
authors.forename: N
authors.initials: N
authors.lastname: Fretwell
authors.forename: A J
authors.initials: AJ
authors.lastname: Martin
authors.forename: P G
authors.initials: PG
authors.lastname: Jones
authors.forename: C A
authors.initials: CA
authors.lastname: Jones
authors.forename: W E R
authors.initials: WE
authors.lastname: Ollier
mesh_terms: D000483:Alleles; D000818:Animals; D004285:Dogs / Q000097:blood / Q000235:genetics*; D005455:Fluorescent Antibody Technique / Q000379:methods; D015395:Histocompatibility Antigens Class I / Q000032:analysis* / Q000235:genetics; D006650:Histocompatibility Testing / Q000379:methods / Q000662:veterinary*; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D015395:Histocompatibility Antigens Class I; D009419:Nerve Tissue Proteins; C467044:RBM45 protein, human; D016601:RNA-Binding Proteins; C039692:histocompatibility antigen DLA