SCA17 Allele Expansion Analysis

Spinocerebellar ataxia type 17 allele CAG/CAA-repeat tracts from 30 patients from this cohort were PCR amplified using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers which flank the CAG/CAA-repeat region and introduce restriction enzyme cleavage sites. The primers used were TBPx3 BamHI Forward (5′-ATGTTTggatccTCCACAGGGTGCCATGAC-3′) and TBPx3 XhoI Reverse (5′-GGTTTGctcgagACACGAAGTACTCACTGC-3′), with restriction sites indicated in lower case. The PCR reactions were processed and cloned into a pcDNA3.1(+) vector similar to previously described for SCA1 alleles (Menon et al., 2013 (link)) or pCR-Blunt using the Zero Blunt PCR Cloning Kit (Thermo Fisher Scientific). Clone plasmids were propagated in Stbl3 E. coli (Thermo Fisher Scientific) (genotype F⁻mcrB mrr hsdS20(r_B⁻, m_B⁻) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str^R) xyl-5 λ⁻leumtl-1). Plasmids were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific).