Fig. 6

Mapping of the LRMD mutation. a Nested RT-PCR on the DMD cDNA. Normal-sized amplicons were obtained from the cDNA isolated from LRMD dogs, including the amplicons obtained using primers for exons 10 and 20 (expected size 1373 bp) and exons 21 and 26 (expected size 807 bp). A RT-PCR using primers to amplify exons 15 to 22 yielded a normal-sized amplicon (1095 bp) in the WT dog, but no band in the LRMD dog. b Southern blot on the EcoRI digested gDNA from two WT Labrador dogs unrelated to the LRMD colony and two LRMD dogs. The EcoRI restriction map of this region is schematized below the southern blot. The digestion profile of samples obtained for two LRMD dogs revealed by Probe 1, covering exons 18-24, differed from the one obtained from healthy animals. In the LRMD restriction map, an extra-band, around 13 kb, and a 2.5 kb band replacing the 2.4 kb wild-type band were observed. The use of Probe 2 covering exon 21 showed an abnormal band at 13 kb that had shifted from its normal size at 10 kb. The use of Probe 3 covering exon 20 showed an abnormal band at 2.5 kb; this band had shifted from its normal size at 2.4 kb. These results suggest that a gross rearrangement in intron 20 had occurred. c Exploration of intron 20 (4.5 kb in WT dogs) in a LRMD and a WT littermate by PCR, using 4 overlapping reactions. Normal size bands were obtained using the first three pairs of primers. The last pair of primers, designed to explore a 1661 bp region including the junction between intron 20 and exon 21, downstream the EcoRI restriction site, failed to amplify any product in LRMD dogs, even when using long-range PCR conditions