Supplemental Figure 3 Female premutation samples digested with

Supplemental Figure 3. Female premutation samples digested with either Hpa. II or Eag. I reveal novel skewing patterns in characteristic female premutation alleles. m. PCR-CE analysis was applied to three samples following either standard digestion with Hpa. II (left panels) or a modified procedure using Eag. I (right panels) for three samples: A) NA 06905, a female premutation with 23, 78 CGG; B) #95, a female premutation with 32, 70 (71 -75) CGG; and C) #76, a female premutation with 32, 107 (109 -125) CGG. The HEX and FAM signals were combined within each electropherogram to contrast differences between the two restriction enzymes. DNA digested with Eag. I was amplified with a forward primer adjacent to this restriction site yielding PCR products 120 bp longer than Hpa. II digested products. A PCR product using the primers designed for the Eag. I site was diluted to 1500 copies and included as reference (“Ctrl”) in each Eag. I-treated reaction. Note that longer PCR products following Eag. I treatment demonstrated lower resolution than the shorter Hpa. II digested products. Two populations of differentially methylated premutation alleles were observed in the two clinical samples but not in the cell line DNA, NA 06905. Results for specific methylation patterns in the premutation allele were consistent between Hpa. II and Eag. I digestions.

Suppl Fig. 3 Hpa. II Digested + Control A) NA 06905 Eag. I Digested + Control 78 CGG 23 CGG 78 CGG Ctrl 23 CGG B) #95 70 CGG 32 CGG 107 CGG 109 -125 CGG Ctrl 70 CGG 71 -75 CGG Ctrl 32 CGG Ctrl C) #76 32 CGG 71 -75 CGG 32 CGG Ctrl 107 CGG 109 -125 CGG