Restriction enzyme based analysis of the methylation status at the FOXP3 −77 reporter position.

PCR product from bisulphite treated DNA was digested with the restriction enzyme Aci1, which only cleaves DNA at position −77 in the FOXP3 promoter when DNA originally was methylated. Digestion was followed by GeneScan analysis. (A) Electropherograms showing peaks from the Aci1 digestion product. Amounts of digested and undigested product were calculated as area under the curve. Shown are results from one representative donor out of three analyzed, except for electropherogram obtained from CD19⁺ cells where one single donor was analyzed. Digestion product from methylated and unmethylated BAC was analyzed three times with similar results. (B) Filled bars indicate percentage methylated, and open bars percentage unmethylated −77 sites. Methylase treated and unmodified BACs were used as controls.