Department of Physics, Chemistry and Biology (IFM)

DNA was extracted from chicken brain tissue, both cerebellum and diencephalon (hypothalamus-thalamus), and from a pool of all individuals samples of negative (unmethylated) and positive (fully methylated)  controls were prepared. These controls were then used for calibration of primer annealing temperatures as well as an approximation of the degree of methylation.

Unmethylated cytosines were converted into to tyrosine using bisulfite treatment.. All individual samples, as well as the controls were treated. This treatment generate a sequence difference in cases were individuals have different methylation patterns.

The samples were then analyzed using high-resolution melting, HRM. By melting DNA, sequence differences inherent in their genetic code can be detected as the DNA strands melt at different temperatures if there was a methylation difference before the bisulfite treatment. Simply put, methylated DNA will have kept their cytosine bases, and in the DNA double helix, C-G basepairing have stronger bonds than A-T basepairs. Before melting the DNA, probes that bind double-stranded DNA was added to all samples, and these probes emit fluorescence when they bind. As the temperature increases and the double-stranded DNA becomes single stranded, the fluorescence intensity will drop. We could then compare fluorescence values at set temperatures to investigate differences between individuals.