Reliable sex identification of dogs by modified PCR/RFLP analysis

To find definitive RFLP sites for canine sex determination, DNA segments corresponding to parts of the canine ZFX and ZFY genes were amplified by PCR and were directly sequenced. According to the newly defined sequence data, the combination of HaeIII and Cfr131 sites was found to be useful for not only identifying the sex of the canine DNA samples but also distinguishing them from the human DNA. Conveniently, these two enzymes worked simultaneously in the same single buffer. The double-digestion of the ZFX/ZFY PCR products with HaeIII and Cfr131 showed banding patterns unique to males and females in Canis familialis. This PCR/RFLP method was confirmed to be applicable to various breeds of dog.