HIGH-RESOLUTION COMPARATIVE HYBRIDIZATION TO COMBED DNA FIBERS

Comparative genomic hybridization (CGH) has proven to be a comprehensi ve new tool to detect genetic imbalances in genomic DNA. However, the resolution of this method carried out on normal human metaphase spread s is limited to low copy number gains and losses of greater than or eq ual to 10 Mb. An improved resolution allowing the detection of copy nu mber representations of single genes would strongly enhance the applic ability of CGH as a diagnostic and research tool. This goal may be ach ieved when metaphase chromosomes are replaced by an array of target DN As representing the genes of interest. To explore the feasibility of s uch a development in a model system we used cosmid MA2B3, which encomp asses about 35 kb in the vicinity of exon 48 of the human dystrophin g ene. Linearized cosmid fibers were attached to a glass surface and ali gned in parallel by ''molecular combing''. Two-color fluorescence in s itu suppression hybridization was performed on these cosmid fibers wit h probe mixtures containing different ratios (ranging from 1:2 to 4:1) of biotin- and digoxigenin-labeled MA2B3 cosmid DNAs. For each mixtur e fluorescence ratios were determined for 40-50 individual combed DNA molecules. In two series comprising a total of 651 molecules the media n fluorescence ratio measurements revealed a linear relationship with the chosen probe ratios. Our study demonstrates that fluorescence rati o measurements on single DNA molecules can be performed successfully.