Figure 3. .

Compound heterozygous mutations in DSG2 for individual B.II-1. Total RNA was isolated from Epstein-Barr virus–transformed lymphoblasts from individual B.II-1 (Trizol [Invitrogen]; RNeasy clean-up [Qiagen]), and cDNA was generated by reverse transcription (Superscript III 1st-strand cDNA synthesis kit [Invitrogen]). PCR products amplified with primers spanning the proband’s two mutations were cloned into the pCR2.1 plasmid (TOPO-TA cloning kit [Invitrogen]), and several individual clones were sequenced using both M13 forward and reverse primers. Representative chromatograms from two clones are displayed, which demonstrate that the R48H and W305X mutations lie on different alleles.