Optimization of DNA extraction from feathers for PCR-based avian sexing test

DNA extraction is a technique used to isolate DNA. Many avian species are monomorphic and this causes difficulty in breeding avian species for bird breeders due to unsuccessful breading. DNA extraction is a crucial part in molecular sexing thus a reliable technique should be developed. Although several DNA extraction methods have been described, the complexity and laborious procedure has hindered the process. Time effective is always the aim in molecular biology. The purpose of this study is to develop a rapid and efficient mean of DNA sexing. Another aim of this study is to develop a standard operating procedure of avian sex determination in UMK laboratory set up. This is achieved by amplifying chromo-helicase-DNA binding (CHD) genes that are preserved within avian Z and W sex chromosomes where female is heterogametic (ZW) while male is homogametic (ZZ). In this study, genetic source in feather which contains amplifiable DNA is used. Phenol chloroform method is adopted and optimized to develop a reliable and rapid DNA extraction. Polymerase chain reaction (PCR) is applied to amplify a conserved region in CHD gene by P2 and P8 primers. The intron length differences between the two sex chromosomes show single (male) and double (female) bands in gel electrophoresis with approximately 450bp of W fragment and 350bp of Z fragment. The result shows that the extraction process can be done rapidly as compared to the protocol proposed in previous study. The findings of this study show that the minimum concentration and purity needed for a reliable PCR result is 8ng/µL and 1.83. A minimum of 4mg of feather calamus is required to meet the minimum concentration required for a successful PCR result. The findings of this study set a guideline for screening process and standard operating procedure for avian sex determination.