PCR Amplification of Clock and Adcyap1 genes with EmeraldAmp® GT PCR Master Mix in Avian species for polymorphism elucidation

This PCR protocol is used to amplify Clock and Adcyap1 gene regions in avian species which have previously shown polymorphisms, such as poly-Q runs, that correlated to migration phenology. It was tested and optimized in Woodlands kingfisher (Halcyon senegalensis) and Diederik cuckoos (Chrysococcyx caprius). The primers were designed based on those previously used by Johnson et al. (2007) and Steinmeyer et al. (2009) by comparing the relevant gene sequences for chickens (Galus galus) with several other available avian species to select primers that would account for the most common variations in primer regions, enabling more universal amplification. Individual clock gene sequences were retrieved from Genbank and aligned in BioEdit 7.2. Primers were then selected based on the annotated regions. The assay was designed using 25 μL (half) reactions of EmeraldAmp® GT PCR Master Mix, which is premixed with loading buffer for easy gel loading following PCR and does not require a long initial denaturation step (thereby shortening the run time). Gel electrophoresis was able to confirm successful amplification of a product ±280 bp long in both species. The same primers were subsequently used for sanger sequencing. A BLAST search of the resulting sequences confirmed the identity of the amplified regions.