The effect of the intron 3 G→A mutation on RNA splicing. To evaluate the effect of the intron 3 G→A mutation on RNA splicing, we transfected minigene expression vectors into HEK293 cells. Total RNA was purified, which was subjected to RT-PCR analysis. (A) Primer sets for the PCR analysis are shown. Set 1 amplified exon 3 (158 bp), whereas set 2 amplified exon 3 and exon 4 (248 bp). (B) Results of RT-PCR. Primer set 2 yielded the 248-bp band from WT-transfected cells (lane 6), and primer set 2 yielded different products from intron 3 G→A mutant-transfected cells (lane 7). (C) Sequence analysis of the transcript from intron 3 G→A mutant-transfected cells. Underlines indicate the position of the G→A mutation. Forty-nine clones were analyzed: 38 clones had a 105-bp insert, and 11 clones had a 27-bp insert. Each variant was spliced with recognition at the GT-AG motif.