Fig. 4

PCR amplification of 9 short intron regions in DMD mRNA. a Nine short amplified products. Electrophoregrams showing amplification of nine introns, using primers for upstream and downstream exons. Amplifications of regions covering introns 10, 14, 24, 31, 35 and 75 each yielded one band, corresponding to normally spliced products. Amplification of regions covering introns 40, 58 and 70, however, each yielded multiple bands. Amplification of intron 40 yielded three bands. The densest band included a 568-bp fragment of intron 40 (exon 41e) between exons 40 and 41 (*2); the next dense band corresponded to the normally spliced product, consisting of exons 40 and 41; and the largest band contained the full-length of intron 40 between exons 40 and 41 (*1). Amplification of intron 58 yielded three bands. The largest was non-specific; the middle band contained intron 58 between exons 58 and 59 (*3), and the smallest corresponded to the normally spliced product. Amplification of intron 70 yielded two bands, the major band being the normally spliced product, and the larger band containing intron 70 between exons 70 and 71 (*4). The structures of intron retaining products are shown schematically on the right. Boxes and numbers in the boxes represent exons and exon numbers, respectively. Bars indicate introns. b Quantification of amplified products. The peak height of each product was determined semi-quantitatively by the Bioanalyzer, and the percentage of each product relative to all amplified products (vertical bars) was calculated. Clear and shaded boxes represent normal and intron retained products, respectively. The black box represents the product retaining part of intron 40. c Accumulated percentage of three retained introns. The percentage of products with retained introns 40, 58 and 70 is represented by open boxes. Half of the product containing intron 70 lacked exon 70, reducing by half the percentage determined by intron specific PCR. The accumulated percentage was close to 90%. Numbers in the box indicate intron number