HIGH-RESOLUTION DNA FIBER-FISH FOR GENOMIC DNA MAPPING AND COLOR BAR-CODING OF LARGE GENES

We have applied two-colour fluorescence in situ hybridization (FISH) to DNA fibers and combined it with digital imaging microscopy for the mapping of large cosmid contigs. The technique was validated using a set of unique plasmids and a cosmid contig both originating from the thyroglobulin (Tg) gene and previously mapped by restriction analysis. The resolution proved to be close to the theoretical lower limit of similar to 1 kb, ranging greater than or equal to 400 kb. Subsequently a 400 kb cosmid contig derived from a DMD-YAC was directly mapped by Fiber-FISH, The resulting map is in full agreement with the restriction map. Two-colour Fiber-FISH mapping thus showed to be capable for accurately sizing gaps and overlaps, and to identify chimeric or repeat sequence containing cosmids across a 400 kb region at once. The generated 400 kb 'colour bar-code' was subsequently used to map two DMD deletion breakpoints in patient DNA with an accuracy of 1-2 kb, The results underscore the value of this method for the delineation of chromosomal rearrangements for positional cloning and single patient clinical studies.