Figure 2.

Analysis of patient's genomic DNA. A, Southern blot analysis, with a probe for COL5A1 exons 5 and 6. Genomic DNA samples from the patient (P) and from the control individual (C) were cleaved with BamHI (B) or Pst I (P) and were probed for exon 5 and 6 sequences. B, Summary of features in the COL5A1 region affected in the patient. Exons are denoted by vertical bars or unblackened boxes. The exons are connected by horizontal lines that depict introns drawn approximately to scale. Arrowheads denote the positions of PCR primers. A circled P marks the site of a polymorphic PstI site in exon 5 of the patient's allele A. This polymorphic site contributes to the size differences between the patient’s allele A and allele B fragments and is also used as a marker in figure 5. Here and in figure 5, the control individual is also seen to be heterozygous for the PstI site. The obligatory ag dinucleotide of the intron 4 splice-acceptor site is shown for allele A. In allele B, an A→G transition (marked with an asterisk [*]) converts this dinucleotide ag to gg (IVS4-2A→G). Two cryptic splice-acceptor sites used by some allele B RNA transcripts are boxed within a 17-nt sequence shown for the allele B intron 4/exon 5 junction; intron sequences are denoted by lowercase letters, and exon sequences are denoted by uppercase letters. Restriction sites in the upper section of the panel were derived elsewhere (Takahara et al. 1995). Underlined restriction sites in the middle section of the panel were inferred from the Southern blot shown here and from another Southern blot not shown; all other restriction sites in the middle section of the panel were derived from complete sequencing of amplification products generated using primers P1 and P2. A schematic in the bottom section of the panel illustrates relationships between the restriction fragments seen in panel A and various features of the region of COL5A1 affected in the patient. B = BamHI; E = EcoRI; P = PstI; S = SacI; X = XhoI.