Rapid and Simple Mutation Screening of GM1 Gangliosidosis in Shiba Dogs by Direct Amplification of Deoxyribonucleic Acid from Various Forms of Canine Whole-Blood Specimens

Abstract

This report describes a rapid and simple method for mutation screening of GM1 gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine β-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of GM1 gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.