Quantitative DNA fiber mapping

Abstract

High resolution physical maps are indispensable for large-scale, cost-effective gene discovery. The construction of such maps of the human genome and model organisms is one of the major goals of the human genome project. The precise localization of cloned DNA fragments within much larger genomic fragments and knowledge of the extent of overlap between two clones are needed to assemble high resolution physical maps. The fluorescence in situ hybridization (FISH) provides this critical information. Isolation of DNA from cell nuclei and preparation of some sort of DNA “fibers,” that is, chromatin or 30-nm fibers representing bundles of DNA improves accessibility of the DNA target for probes and detection reagents and thus increases the hybridization efficiency. FISH applied to most preparations of decondensed nuclear or isolated cloned DNA allows visualization of probe overlap and provides some information of the existence and size of gaps between two clones. In quantitative DNA fiber mapping (QDFM), a solution of purified DNA molecules is placed on a flat surface prepared so that the DNA molecules slowly attach at one or both ends. Because of the high hybridization efficiency obtained with DNA fibers, QDFM is also the method of choice to optically map expressed sequences in limited genomic intervals.