SCN9A Loss of Function Mutations E1683 Supp. Figure S4. The c.2687G>A mutation does not alter splicing. (a) Partial sequence chromatogram of the RT-PCR spanning exons 14 to 16. The mutation is shown by an arrow. The nucleotides at the normal splicing sites at both sides of exon 15 are boxed. (b) Minigene assay to investigate whether the c.2687G>A mutation disrupts splicing control of SCN9A. The reporter gene is composed of α-globin (black boxes) and fibronectin exons (shaded boxes). WT or mutant coding exon 15 along with upstream and downstream intronic sequence were cloned into the NdeI site of the PTB minigene construct followed by transfection into HeLa cells. RT-PCR analysis shows that in the wild-type context (Lane 1) a PCR product of 594 bp was amplified which represented normal inclusion of exon 15 in the mature transcript. The minigene carrying the mutation behaved in an identical manner (Lane 2).