Figure 6

Application of the method to a HNPCC patient with a PSC. A: BTH102 were transformed with pREAL carrying a msh2 insert obtained from a heterozygous patient. The proportion of red/white colonies was 51.7% and 48.3% respectively. B: Confirmation of the genotype of the different colonies by PCR and RFLP resolved on 10% non-denaturing PAGE. Lane 1 shows the 86bp PCR product of a red colony, without enzymatic incubation. Lane 2: PCR product obtained from a red colony, with subsequent enzymatic incubation. The 67 bp band results from the complete cleavage of the wild type allele in two smaller fragments of 67 bp and 19 bp (band not shown). Lane 3: PCR product obtained from a white colony, without enzymatic incubation. Lane 4: PCR product obtained from a white colony, with subsequent enzymatic incubation. The 86bp band in the mutated allele is not recognized by HaeIII and is not cleaved. C: Western blot analysis of red and white colonies. In lanes 1 and 2, controls for fragment T18 and fragment T25-18 are shown. In lane 3, the western blot on the red colony reveals the presence of the complete adenylate cyclase, with the wild type version of the msh2 fragment. Notice two re-initiation events: one that corresponds to fragment T18, and a larger band, that corresponds to the presence of an ATG codon in the insert. In lane 4, the Western blot of the white colony confirms the absence of full-lenght adenylate cyclase, and reveals the same re-initiation events seen in the red colonies, one corresponding to T18 and another corresponding to an ATG post PSC.