Exon Trap Analysis
DPAGT1 exons 2, 3 and 4 and flanking intronic sequences or exons 6, 7 and 8 and flanking sequences were cloned into the pET01 vector (MoBiTec). c.478G>A and c.791T>G were respectively introduced by site-directed mutagenesis using Quikchange kit from Stratagene and confirmed by Sanger sequencing. Control and mutant vector DNA were electroporated into the human rhabdomyosarcoma cell line TE671 using the NEON electroporator (Invitrogen). Total RNA was purified 48 hr after transfection, reverse transcribed into cDNA using Retroscript kit (Ambion). cDNA was amplified using primers specific to the vector exons. The amplicons were run on agarose/TBE gels, visualized under UV/ethidium bromide and then gel purified and sequenced.