title: High-resolution comparative hybridization to combed DNA fibers.
abstract: Comparative genomic hybridization (CGH) has proven to be a comprehensive new tool to detect genetic imbalances in genomic DNA. However, the resolution of this method carried out on normal human metaphase spreads is limited to low copy number gains and losses of > or = 10 Mb. An improved resolution allowing the detection of copy number representations of single genes would strongly enhance the applicability of CGH as a diagnostic and research tool. This goal may be achieved when metaphase chromosomes are replaced by an array of target DNAs representing the genes of interest. To explore the feasibility of such a development in a model system we used cosmid MA2B3, which encompasses about 35 kb in the vicinity of exon 48 of the human dystrophin gene. Linearized cosmid fibers were attached to a glass surface and aligned in parallel by "molecular combing". Two-color fluorescence in situ suppression hybridization was performed on these cosmid fibers with probe mixtures containing different ratios (ranging from 1:2 to 4:1) of biotin- and digoxigenin-labeled MA2B3 cosmid DNAs. For each mixture fluorescence ratios were determined for 40-50 individual combed DNA molecules. In two series comprising a total of 651 molecules the median fluorescence ratio measurements revealed a linear relationship with the chosen probe ratios. Our study demonstrates that fluorescence ratio measurements on single DNA molecules can be performed successfully.
authors.affiliation: Institut für Humangenetik, Ruprecht-Karls Universität Heidelberg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Kraus
authors.forename: R G
authors.initials: RG
authors.lastname: Weber
authors.forename: M
authors.initials: M
authors.lastname: Cremer
authors.forename: T
authors.initials: T
authors.lastname: Seebacher
authors.forename: C
authors.initials: C
authors.lastname: Fischer
authors.forename: C
authors.initials: C
authors.lastname: Schurra
authors.forename: A
authors.initials: A
authors.lastname: Jauch
authors.forename: P
authors.initials: P
authors.lastname: Lichter
authors.forename: A
authors.initials: A
authors.lastname: Bensimon
authors.forename: T
authors.initials: T
authors.lastname: Cremer
mesh_terms: D001710:Biotin; D003360:Cosmids; D004247:DNA / Q000378:metabolism*; D004076:Digoxigenin; D016189:Dystrophin / Q000235:genetics; D005240:Feasibility Studies; D018628:Gene Dosage; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization / Q000379:methods
chemical_list: D016189:Dystrophin; D001710:Biotin; D004247:DNA; D004076:Digoxigenin