Genotyping Shorthorn cattle for generalised glycogenosis

Objective To develop a procedure for routine genotyping of Shorthorn cattle for the generalised glycogenosis allele in exon 18 of the acidic alpha -gl ucosidase gene. Procedure Allele-specific amplification and double mismatch amplification p rocedures for the discrimination of the exon 18 alleles were evaluated usin g leucocytes and hair roots as sources of target DNA. Results Allele-specific amplification was effective for genotyping Shorthor n cattle at the 2454 site when purified DNA was used as target for the poly merase chain reaction. However, when the target DNA was derived from hair r oots, differences in the relative yield of wild-type and mutant amplicons w ere observed. The double mismatch amplification procedure was effective in genotyping all subjects, independent of the source of DNA. The unique cleav age sites for Drd I and PshA I within exon 18 are present and absent respec tively in the wildtype amplicon, and are lost and acquired, respectively, i n the mutant amplicon. In addition, the Drd I and PshA I mismatching cleava ge sites incorporated into the primers serve as internal controls for Drd I and PshA I cleavage. Conclusion The double Drd I/PshA I mismatch amplification procedure using h air root samples as the source of DNA is a robust method for genotyping Sho rthorns for generalised glycogenosis.