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.