Bisulfite sequencing of DNA from brain tissue punches. DNA (100 ng) from PVN tissue punches (n = 5) were separately bisulfite treated. Bisulfite primers directed against the mouse Zac1 differentially methylated region (DMR) were used in a PCR. The amplified products were then cloned and DNA from 30 separate recombinant colonies sequenced. Data analysis revealed that the Zac1 locus is methylated at levels of 52% ± 5% corresponding to current literature that the gene is maternally imprinted.