Standardization of a PCR-HRM assay for DNA sexing of birds
Sex determination of birds is of great significance in many ecological and demographic studies, where sex ratio between sexes is important. Obtaining this information is difficult in many avian species since more than 50% of all bird species are sexually monomorphic. This presents a challenge for avian breeders and evolutionary studies since sex identification is vital in the field. The problems associated with accurate avian gender determination in the field were conquered by the introduction of molecular techniques through the knowledge of sex linked genes. This molecular-based technique widely applied for sexing birds is based on intronic variation of the Chromo-helicase-DNA-binding (CHD) gene on the Z and W chromosomes. The intronic length variations between the CHD-Z and CHD-W resulted in the development of a polymerase chain reaction (PCR) approach that allow for sex discrimination. However, the classic PCR-based techniques are time consuming and laborious as they involve a minimum of three steps: DNA isolation, PCR and gel electrophoresis. In this study a PCR-high resolution melt (HRM) assay was developed for the molecular gender identification of avian species. The HRM protocol for avian molecular discrimination was developed based on the difference in melting curve patterns of the CHD1 fragments. The new test was then compared to the conventional PCR assay using numerous different bird species. Although the PCR-HRM assay performed well in Lovebird species, the assay showed poor PCR amplification in other species, which directly affected the accuracy of the HRM technique in these species. Therefore, the newly established PCR-HRM assay could not be recommended for implementation in a commercial DNA diagnostics laboratory.