title: Two novel real-time PCR methods for genotyping the von Willebrand disease type I mutation in Doberman Pinscher dogs.
abstract: 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 ≥ 6.4 ng genomic DNA per reaction and the MGB real-time PCR assay yielded reliable results with ≥ 150 pg 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.
authors.affiliation: Department of Veterinary Medical Sciences, Alma Mater Studiorum, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano dell'Emilia, Bologna, Italy. fabio.gentilini@unibo.it
authors.forename: Fabio
authors.initials: F
authors.lastname: Gentilini
authors.forename: Maria E
authors.initials: ME
authors.lastname: Turba
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000235:genetics*; D004285:Dogs; D020022:Genetic Predisposition to Disease; D005838:Genotype; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D014842:von Willebrand Diseases / Q000235:genetics / Q000662:veterinary*
keywords: Canine; Genotyping; Polymorphism; Single tube real-time PCR; von Willebrand disease type I