PCR-SSCP analysis of the ovine DQA2 locus: A dissertation submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours at Lincoln University

Abstract

Strong association between RFLPs in certain OLA DQA2 alleles and footrot resistance has been demonstrated (Escayg, Ph.D. thesis, 1995). The use RFLP-Southern hybridisation analysis as a routine DQA2 typing assay is limited by technical and practical inefficiencies. In the present study, PCR-SSCP analysis, with silver staining, was adapted to allow its application to the DQA2 locus. The three aims of this investigation were; to demonstrate the ability of SSCP to reveal greater DQA2 polymorphism than did RFLP, to identify homozygotes for sequence analysis,establish worth of SSCP as a routine typing system. Analysis of homozygous samples produced results which strongly suggested greater diversity exists, with DQA2 CC animals showing low PCR amplification and complex SSCP banding patterns. Uncertainty created by elaborate banding patterns, inefficient silver staining, and non-DQA2-specific PCR primers prevented confident identification of homozygotes for sequencing. Sequence information is necessary, and a different approach is required. SSCP analysis of heterozygotes demonstrated the ability of SSCP to resolve distinct DQA2 genotypes. Distinct banding patterns characteristic of individual alleles could not be defined. This is required if PCR-SSCP is to provide a routine DQA2 typing assay, and much persistence may be required. Sequence information may be the most direct route to establishing a molecular understanding of variation in footrot resistance. However, technical limitations of SSCP analysis need to be addressed, and the critical gentic loci confirmed, before expenditure can be justified. Low numbers of homozygotes may reflect MHC-mediated infertility caused by inbreeding.