Quantitative DNA Fiber Mapping

Several hybridization-based methods that are used to delineate single-copy or repeated DNA sequences over larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound to a solid substrate at one end (at least). The DNA molecules are then stretched by the action of a receding meniscus at the water–air interface, which results in the DNA molecules being stretched homogeneously to about 2.3 kb/µm. When nonisotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, and their respective binding sites are visualized under the fluorescence microscope, their relative distances can be measured and converted into kilobasepairs (kb). The QDFM technique has found a variety of useful applications, ranging from the detection and delineation of deletions or overlaps between linked clones, to the construction of high-resolution physical maps and studies of stalled DNA replication and transcription.