Reliable sex identification of dogs by modified PCR/RFLP analysis

To find definitive RFLP sites for canine sex determination, DNA segments co rresponding 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 o nly identifying the sex of the canine DNA samples but also distinguishing t hem from the human DNA. Conveniently, these two enzymes worked simultaneous ly in the same single buffer. The double-digestion of the ZFX/ZFY PCR produ cts with HaeIII and Cfr131 showed banding patterns unique to males and fema les in Canis familialis. This PCR/RFLP method was confirmed to be applicabl e to various breeds of dog.