Journal article
Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population
Immunogenetics (New York), Vol.62(9), pp.633-639
09/2010
Handle:
https://hdl.handle.net/2376/105514
PMCID: PMC3359745
PMID: 20683590
Abstract
Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (positive control), and random nucleotide (negative control) 23- to 27-mer oligonucleotide microarray probes were designed and spotted onto an epoxy-coated masked slide using a robotic arrayer. Bulk RT-PCR products from each horse were biotinylated by nick translation, hybridized to the array, and detected using tyramide signal amplification. The microarray consistently detected eight of nine classical MHC class I transcripts and allowed ELA haplotypic associations to be made. Cloning and sequencing of RT-PCR products were then performed in a group of ELA disparate horses and ponies, in which six novel sequences were identified. This group was used to determine the specificity of the array. Overall, the microarray was more efficient than cloning and sequencing for detecting expressed classical MHC class I sequences in this defined population of horses, and was significantly more specific than serology. These results confirmed the utility of a microarray-based method for high-resolution MHC class I typing in the horse. With additional probes the array could be useful in a broader population.
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Details
- Title
- Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population
- Creators
- Joshua D Ramsay - Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USASteven R LeibLisa OrfeDouglas R CallRebecca L TallmadgeDarrilyn G FraserRobert H Mealey
- Publication Details
- Immunogenetics (New York), Vol.62(9), pp.633-639
- Academic Unit
- Veterinary Clinical Sciences, Department of; Veterinary Microbiology and Pathology, Department of; Paul G. Allen School for Global Animal Health
- Publisher
- United States
- Grant note
- K02 AI073101 / NIAID NIH HHS AI073101 / NIAID NIH HHS
- Identifiers
- 99900546750501842
- Language
- English
- Resource Type
- Journal article