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.