Gender identification in Chicken (Gallus gallus) by PCR using whole blood and dried blood spot on filter paper as template: without prior DNA isolation

Abstract

Accurate sex identification of pure line chickens in their early age has significant economic impact in breeding industry. In the recent years, range of Polymerase Chain Reaction (PCR) based sex determination techniques are routinely used to identify the sex of parent lines in breeding industries, however purified DNA is a prerequisite. Hence this study was aimed to develop a rapid and inexpensive PCR based gender identification method for chicken using whole blood samples and dried blood spots as template for PCR without DNA extraction. In addition, practicability of two W-chromosome specific gene targets in chicken for sex determination also characterised. Successful amplification of sex specific fragments and an internal control was achieved with the range of 0.125l and 0.250l volume of whole blood on filter paper (~1 mm) prepared from chicken and dried blood spot. This technique does not require DNA extraction, freeze/thawing of blood samples, pre-treatment with any reagents, dilution of whole blood or dried blood spots on filter paper. It can be carried out with commercially available Taq polymerase enzymes with increased concentration of MgCl2 (3 mM) and 0.5% of DMSO without optimisation of PCR buffers. In conclusion, as compared to the existing PCR based sex identification techniques, the present approach is relatively economic, time saving, requires minimal steps and eliminates the need for DNA extraction.