Simple and rapid genotyping of D-amino acid oxidase gene recognizing a crucial variant in the ddY strain using microchip electrophoresis

A rapid genotyping method of D-amino acid oxidase (DAO), an enzyme that catalyzes the oxidative degradation of most of the D-amino acids in mammals, has been established. This method employs a one-step PCR, restriction enzyme digestion and rapid microchip electrophoresis (MCE), and the DAO genotype of the living individual mice was definitely determined within a day by clearly separating the 95 and 107 bp Hpa II digested DNA fragments. For verification of the method, the DAO activity in the kidney of individual mice was also determined, and the obtained values completely matched the estimated genotypes (DAO^+/+, DAO^+/-, and DAO^-/-). The intrinsic amounts of D-Pro in the serum and kidney of mice with three DAO genotypes were compared for the first time, and demonstrated that the D-amino acid amounts in the DAO^+/+ mice (1.93 ± 0.66 nmol/mL serum, not detectable in the kidney) and DAO^+/- mice (1.50 ± 0.24 nmol/mL serum, not detectable in the kidney) were almost the same. The present method should be a powerful tool to establish various pathologic-model animals under the complete care of their intrinsic DAO activity, which are useful for the screening of D-amino acids having physiological activity and/or diagnostic values.