Supplemental Figure 4. Gene-specific FMR1 PCR Enables Detection of Low Abundance Expanded Alleles. A) FMR1 Southern blot data for 5 clinical specimens. The threshold for full mutation (FM, >200 CGG) unmethylated and methylated alleles is designated by the black dotted line. Note that the expanded alleles represented in samples #118 and #125 were very faint, but these were identified as full mutations. B) Corresponding FMR1 PCR data, as visualized by AGE. The threshold for a full mutation (FM) is indicated by the white dotted line. C) Corresponding FMR1 PCR data, as visualized by CE. The threshold for a full mutation (FM) is marked with the black vertical line. To facilitate data interpretation, the Y-axis scale was set to best suit the allele representations for each specimen: #22 (150 RFU), #101 (200 RFU), #63 (1000 RFU), #118 (1100 RFU), and #125 (1100 RFU). Note that the full mutations in #118 and #125 are readily discerned. The same gDNA sample was used for all three evaluations (Southern blot, PCR/AGE, and PCR/CE).

Fm, Full Mutation Mosaic

#22 #101 #63 #118 #125

Sou

ther

n B

lot

Dat

aP

CR

/AG

E D

ata

Southern PM PM FM FM FMCall

PCR Call Fm Fm Fm Fm Fm

PCR/CE Data

#22

#101

#63

#118

#125

Normal Premutation Full Mutation Int

FMMe

FMUnMe

FM

A C

B