Large minigene enables accurate RNA analysis of the ABCA4 noncanonical splice site variant c.5714+5G>A. (A) Minigene containing the genomic region encompassing ABCA4 exon 40 (MG_ex40, black rectangle) and flanked by rhodopsin (RHO) exons 3 and 5 (open boxes) was designed to investigate the effect of the noncanonical splice site variant c.5714+5G>A. Reverse-transcription polymerase chain reaction (RT-PCR) performed using primers (open arrowheads) targeting RHO exons 3 and 5 showed an expected 404-bp wild-type fragment and a 274-bp fragment corresponding to exon 40 skipping (Δ) in control minigene (+5G) and full exon 40 skipping in mutant minigene (+5A). (B) Larger minigene containing the genomic region encompassing ABCA4 exons 39–41 (MG_ex39–41) was designed to investigate the same variant. RT-PCR using primers (black arrowheads) targeting exons 39 and 41 showed a single 260-bp wild-type fragment in the control minigene, while in the mutant minigene fragments of 260 and 130 bp corresponding to wild-type and exon 40 skipping were found.
