DLA-DRB1 histocompatibility genotyping using RT-nested PCR and cycle sequencing.
Class-II histocompatibility genes are associated with predisposition to autoimmune diseases in many mammal species. We have developed a technique using reverse transcriptase and nested-PCR for amplification from blood samples of expressed sequences encoded by canine DLA-DRB1 loci. In the first polymerase chain reaction (PCR), we utilize primers DR-SP and DR-STOP as developed by Sarmiento et al. (1990). In the nested PCR, we utilize two additional primers, namely primer 57 [5'-TCTTGGAGGCTCCTGGATGACAGC-3'] and primer 367 [5'-CACAACTACGGGGTGATTGAGAGC-3'] to produce a 334 bp amplified product. After digestion with restriction endonucleases, some of the alleles can be identified by restriction fragment length polymorphism (RFLP). The increasing information on new DLA-DRB1 alleles over the last two years renders the DLA-DRB1 too diverse for convenient use of RFLP. However, the expressed sequences amplified by our protocol can be conveniently identified by cycle sequencing. This RT n-PCR protocol will suffice for the genotyping of individual dogs at the DLA-DRB1 locus.
Happ
G M
GM
Institute of Arctic Biology, University of Alaska-Fairbanks, 99775, USA.
Aquilla
E
E
Martick
M
M
Yuncker
C
C
Wojciechowski
J
J
Fox
L
L
D000483:Alleles; D000818:Animals; D004285:Dogs / Q000235:genetics* / Q000276:immunology; D005260:Female; D005838:Genotype; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D008297:Male; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012150:Polymorphism, Restriction Fragment Length
D000949:Histocompatibility Antigens Class II