DNA isolation and Small pool PCR analysis
For each participant, sizing of the expanded allele was done twice: 1) in the biopsy tissue from which myoblasts were isolated, and 2) in the same myoblast cultures that were used for FISH staining and RNA isolation. In the biopsy tissue, DNA extraction was performed following the phenol:chloroform:isoamyl alcohol method. In the myoblast cultures, DNA was extracted using a previously described protocol for blood DNA extraction (Miller, Dykes, & Polesky, 1988). To determine expansion size in these DNAs, we first performed a long PCR using the primers DM-C and DM-DR described elsewhere (Gomes-Pereira et al., 2004; Monckton et al., 1995; Salinas-Rios et al., 2011) and PCR Master Mix (Thermo Fisher Scientific; MA). We supplemented the reaction with 69 mM 2-mercaptoethanol, 5% DMSO, and Taq polymerase (Sigma-Aldrich; Gillingham, UK) at 1 unit per 10 µL. The annealing temperature was 63.5°C. The long PCR products were resolved by electrophoresis on a 1% agarose gel and hybridized by Southern Blot as previously described (Gomes-Pereira et al., 2004; Monckton et al., 1995). Autoradiographic images were scanned and the progenitor allele in each patient was estimated by comparison against the molecular weight ladder, using GelAnalyzer 19.1 software.