PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased Japan since the late 2000s. Recently, we demonstrated that JRTs with GI harbor identical germline variant APC gene (c.[462_463delinsTT]) heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although affected have distinct features, such as development multiple and early age onset, genetic testing indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable detecting were designed validated using synthetic wild-type mutant DNAs genomic from carrier non-carrier dogs. Result First, PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage creating new restriction site Mse I. In PCR-RFLP assay, 156-bp region containing amplified PCR subsequently digested I, yielding diagnostic 51 58 bp fragments allele allowing determination genotypes. It possible to determine genotypes DNA extracted peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, TaqMan duplex real-time developed, where 78-bp flanking presence allele- allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles amplification determined all examined samples measuring fluorescence intensities. Importantly, false-positive false-negative errors never detected both assays. Conclusion study, highly reliable tests polyposis JRTs, providing accurate assessment causative variant. genotyping could contribute diagnosis prevention