The identification of species and their sex from small biological samples is of scientific interest in forensic science. Various identification techniques have been developed
however, DNA-based PCR is the most specific and sensitive technique compared to protein-based methods. Although PCR amplification of the amelogenin (AMEL) has been used in different species for sex determination, the reliability of the AMEL test may sometimes be challenged due to amplification failure of AMEL Y in males, resulting in incorrect gender identification. Therefore, this study aimed to establish a simple, reliable and accurate PCR protocol for the amplification of the AMEL gene from blood gDNA isolated by a single-step DNA isolation method using primers of different GC% to ascertain the sex of ovine. This methodology may also be applicable to various biological samples for sex determination. It was concluded that the touchdown PCR was more suitable for GC-rich primers and low GC% primers were suitable with modified conventional PCR for gender identification. The use of PCR enhancers at denaturation temperatures of 94°C and 95°C was found ineffective for the amplification of AMEL to determine the sex. In summary, all primers used showed successful amplification.