Materials and Methods
Clinical DMD MLPA analysis was performed on proband to confirm
the presence of the duplication, assess its size, and confirm a DMD
diagnosis. MLPA experiments included probes for each of the 79DMD coding exons, 3’UTR, 5’UTR, one probe for the alternative
promoter/exon 1 DP427c, as well as control probes (MRC Holland).
Capillary electrophoresis of PCR amplified ligated probe pairs was
performed on ABI 3730xl (Applied Biosystems), and data analyzed with
GeneMarker Software (SoftGenetics LLC, State College, PA, USA). CMA
analysis was performed on proband using CytoScan HD Suite (Thermo Fisher
Scientific, Waltham, MA, USA) according to manufacturer’s instructions.
CMA minimal and maximal probe positions for the duplication were used to
design sequencing primers using Primer3Plus. Forward primer sequence:
ctgtgttttgggccatttct and reverse primer sequence: tgggtttagccctaggacac
produced a ~1.8Kbp product visualized in 2% agarose gel
in a UV light box. The 1.8Kbp product was cleaned up using Exo-SAP-IT
PCR Product Cleanup Reagent (ThermoFisher Scientific, Waltham, MA, USA)
and Sanger sequenced on the ABI 3730xl (Applied Biosystems, Foster City,
CA, USA) according to the manufacturer’s protocol. Chromatogram files
were analyzed in FinchTV (Geospiza, Inc.) and FASTA files exported and
mapped to hg19 using BLAT in the UCSC genome browser [12].