Figure 1.

Methylation-sensitive PCR to validate the candidate regions and sites for tail sexing. In regions (a), the hypermethylated sex has one or more restriction cleavage sites for the methylation-sensitive restriction enzyme HpaII (CCGG) surrounded by methyl groups. In candidate sites (b), a single differentially methylated cytosine is located within the restriction cleavage site. In both these cases (regions and sites), methylation within the restriction sequence prevents DNA digestion by HpaII, while hypomethylation at this site permits DNA digestion. Primers F1 and R1 produce an amplicon irrespective of DNA methylation at the candidate region or site, and thus serve as a control, whereas amplicon 2 is only produced by primers F1 and R2 in the presence of methylation at the restriction site (when DNA digestion is prevented).