Single nucleotide polymorphism analysis
To differentiate DMPK transcript qPCR expression originating from the expanded allele versus the one of the wild type allele, the presence of a previously described informative SNP in exon 10 of DMPK gene (Korneluk, 1993) was studied. If patients with DM1 are informative for the SNP, specific qPCR designed primers could be used for detection ofDMPK expression originating from the expanded allele and specific qPCR designed primers could be used for detection of DMPKexpression originating from the wild type allele.
To evaluate the presence of the aforementioned SNP at the patient DNA level, we used primers 5’-CTGCAGAAGGTTTAGAAAGAGC-3’ (forward) and 5’-CATCCTGTGGGGACACCGAGG-3 (reverse) (Korneluk, 1993) with the following conditions: 94 °C for 30 s, 60 °C for 30 s and 72ºC for 30 s for 30 cycles. Purified products were sequenced with BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster city, CA) using primer forward. Sequences were analyzed with Chromas version 2.6.2., using NG_009784.1 as DMPK reference sequence.