Epsilon Archive for Student Projects

Abstract

This study aimed to develop and evaluate a long-range PCR (LR-PCR) based genotyping system
for the canine major histocompatibility complex (MHC) loci, with a focus on three classical class II
genes. Due to the high polymorphism of the MHC region, common genotyping methods are limited
by both resolution and coverage. A LR-PCR approach was optimized to enable full-length
amplification of DLA-DRB1, DLA-DQA1 and DLA-DQB1, genes, followed by confirmation using
Sanger sequencing.
Optimization experiments assessed Taq polymerase alternatives, primer design, DNA concentration,
and annealing temperatures. Successful amplification of full-length class II genes was achieved.
Sequencing of LR-PCR products was validated against conventional exon 2 PCR and showed
concordant base calls, confirming high accuracy. Allele calling demonstrated high sequence identity
and quality scores above Q20 for all loci. In a heterozygous sample, consistent dual-peak patterns
found by both sequencing methods indicated that LR-PCR preserved allelic variation without bias.
Attempts to include the class I gene DLA-64 were inconclusive due to inconsistent primer specificity
and issues likely stemming from poor reference genome annotation for this locus. Overall, the
developed genotyping system provides a reliable and adaptable method for genotyping highly
polymorphic DLA class II loci, providing a foundation for further development and upscaling with
long read sequencing. While further validation with diverse samples are needed, this study offers a
start for improved canine immunogenetics research, disease association studies, guidelines for
breeding programs and tools for potentially preserving MHC variation in dogs.