Figure 5. Sequencing and RT–PCR analysis of the cultured skin fibroblast mRNA. A: Strategy for amplification of the COL4A5 cDNA by PCR. Exons are represented by open rectangles and are numbered. The localizations of the two amplified fragments are shown with the primer binding sites (horizontal arrows). B: Sequencing of the COL4A5 cDNA amplified by a pair of primers 1/1’ in III-1. The junctions of exons 45, 46, and intron 45 are shown by the vertical lines, respectively. N: normal sequence. C: Left; RT–PCR amplification of a 284 bp fragment of COL4A5 mRNA using a pair of primers 2/2’. M: DNA molecular mass marker; Lane 1 to 4: PCR products of COL4A5 cDNA from III-1, II-2 and two normal male controls, respectively. As compared with the normal individuals, III-1 had a single larger sized RT–PCR product that contained a 71- base- pair indel identified in exon 45, whereas II-2 had two RT–PCR products: the normal sequence and the mutant sequence. C: Right; Schematic representation of the aberrant COL4A5 cDNA resulting from the splice site mutation. Exons are represented by open rectangles and are numbered, and intron 45 is indicated by the horizontal line. D: Forward (Left) and reverse (Right) sequencing of the COL4A5 cDNA amplified by a pair of primers 2/2’ in II-2. The junctions of exons 45 and 46 are shown by the vertical lines, respectively. N: normal sequence.