Thesis
Development of a DNA microarray for detection of expressed equine classical MHC class I alleles in a defined population
Washington State University
Master of Science (MS), Washington State University
2009
Handle:
https://hdl.handle.net/2376/105294
Abstract
Development of an accurate and efficient molecular-based equine major histocompatibility complex (MHC) class I typing method is necessary to advance the study of T lymphocyte immune responses to intracellular pathogens. A DNA microarray is one means by which multiple MHC class I genes can be detected simultaneously and discriminated on the basis of as little as a single nucleotide difference. In this study, expressed classical MHC class I genes from six Arabian horses, homozygous and heterozygous for serologically defined equine leukocyte antigen (ELA)-A1, A4, and W11 haplotypes, plus a serologically undefined haplotype, were amplified by reverse transcription (RT)-PCR using conserved primers, cloned, and sequenced. A total of nine different expressed classical MHC class I alleles were detected, with between two and five alleles identified in each horse. Allele-specific, conserved (positive control), and random nucleotide (negative control) 23- to 27-mer oligonucleotide microarray probes were designed based on these sequences, 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. In all horses it was possible to accurately detect the alleles associated with each haplotype with greater efficiency than cloning and sequencing. The microarray was therefore an effective MHC class I typing method for the horses in this defined population, and will be useful for screening experimental subjects for studies evaluating T cell vaccines and immunotherapeutic strategies against a variety of equine infectious diseases.
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Details
- Title
- Development of a DNA microarray for detection of expressed equine classical MHC class I alleles in a defined population
- Creators
- Joshua David Ramsay
- Contributors
- Robert H. Mealey (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Veterinary Medicine, College of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; Pullman, Wash. :
- Identifiers
- 99900525115601842
- Language
- English
- Resource Type
- Thesis