Australian veterinary journal · 2001
Genotyping shorthorn cattle for generalised glycogenosis.
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Dennis J A, Healy P J
Аннотация
OBJECTIVE: To develop a procedure for routine genotyping of Shorthorn cattle for the generalised glycogenosis allele in exon 18 of the acidic alpha-glucosidase gene.
PROCEDURE: Allele-specific amplification and double mismatch amplification procedures for the discrimination of the exon 18 alleles were evaluated using leucocytes and hair roots as sources of target DNA.
RESULTS: Allele-specific amplification was effective for genotyping Shorthorn cattle at the 2454 site when purified DNA was used as target for the polymerase chain reaction. However, when the target DNA was derived from hair roots, differences in the relative yield of wild-type and mutant amplicons were observed. The double mismatch amplification procedure was effective in genotyping all subjects, independent of the source of DNA. The unique cleavage sites for Drd I and PshA I within exon 18 are present and absent respectively in the wildtype amplicon, and are lost and acquired, respectively, in the mutant amplicon. In addition, the Drd I and PshA I mismatching cleavage 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 hair root samples as the source of DNA is a robust method for genotyping Shorthorns for generalised glycogenosis.
PROCEDURE: Allele-specific amplification and double mismatch amplification procedures for the discrimination of the exon 18 alleles were evaluated using leucocytes and hair roots as sources of target DNA.
RESULTS: Allele-specific amplification was effective for genotyping Shorthorn cattle at the 2454 site when purified DNA was used as target for the polymerase chain reaction. However, when the target DNA was derived from hair roots, differences in the relative yield of wild-type and mutant amplicons were observed. The double mismatch amplification procedure was effective in genotyping all subjects, independent of the source of DNA. The unique cleavage sites for Drd I and PshA I within exon 18 are present and absent respectively in the wildtype amplicon, and are lost and acquired, respectively, in the mutant amplicon. In addition, the Drd I and PshA I mismatching cleavage 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 hair root samples as the source of DNA is a robust method for genotyping Shorthorns for generalised glycogenosis.