Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

摘要

Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificialchromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or thepreparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloningprocess and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged.Some regions of the human genome, however, appear to have a higher propensity than others to rearrange in any hostsystem. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed atechnique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements ofinterest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. Thispaper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, highresolutionphysical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for threedifferent YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstratedunambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was notavailable, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared fromhuman genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitatingclosure of the maps of the human genome as well as those of model organisms.