Two novel real-time PCR methods for genotyping the von Willebrand disease type I mutation in Doberman Pinscher dogs

• Published in: The veterinary journal (1997) Vol. 197; no. 2; pp. 457 - 460
• Main Authors: Gentilini, Fabio, Turba, Maria E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.08.2013
• Subjects: alleles; Animals; blood; blood coagulation factors; Canine; DNA; Doberman Pinscher; Dog Diseases - genetics; Dogs; Genetic Predisposition to Disease; Genotype; Genotyping; mutation; Polymorphism; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - veterinary; Single tube real-time PCR; von Willebrand disease type I; von Willebrand Diseases - genetics; von Willebrand Diseases - veterinary; von Willebrand disease type I; Single tube real-time PCR; Genotyping; Canine; Polymorphism
• ISSN: 1090-0233; 1532-2971
• DOI: 10.1016/j.tvjl.2013.02.023

Two single tube real-time PCR methods were designed to genotype the mutation responsible for von Willebrand disease type I (von Willebrand factor c.7437G>A) in Doberman Pinscher dogs: (1) the Divergent PCR assay, which is a modification of the bi-directional PCR amplification of a specific allele (BI-PASA) technique, and (2) a minor groove binder (MGB) real-time PCR assay using fluorescently labelled probes. There was complete agreement between the genotypes determined using the two real-time PCR methods and the results of sequencing of PCR products generated by conventional PCR from genomic DNA purified from the blood of 27 Doberman Pinscher dogs. The Divergent PCR assay yielded reliable results with ⩾6.4ng genomic DNA per reaction and the MGB real-time PCR assay yielded reliable results with ⩾150pg genomic DNA per reaction. Both real-time PCR methods are suitable for routine genetic testing for the von Willebrand disease type I mutation using blood samples.