Heinz-Ulrich G. Weier, Mei Wang, Jan-Fang Cheng, Yiwen Zhu, Herbert W. Moise, Christopher H. Martin, Micheal J. Palazzolo and Joe W. Gray
Resource for Molecular Cytogenetics and Human Genome Center, Life Sciences Division, 74-157, University of California, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
"Quantitative DNA Fiber Mapping" (QDFM) recently developed in our laboratory combines three techniques (MOLECULAR COMBING, FISH and IMAGE ANALYSIS) for precision mapping of individual DNA fragments and determination of distance or overlap between pairs of cloned DNA fragments.
In MOLECULAR COMBING, a solution of purified DNA molecules is placed on a silanated glass slide prepared so that the DNA molecules slowly attach at one or both ends. The DNA solution is then spread over a larger area by placing a coverslip on top, DNA molecules are allowed to bind to the surface and dried. Individual DNA molecules are straightened and uniformly stretched during drying by the hydrodynamic action of the receding meniscus. The position of specific sequences along the stretched DNA molecules is visualized by overnight fluorescence in situ hybridization (FISH) and measured by digital IMAGE ANALYSIS techniques on images recorded from the fluorescence microscope.
In pilot experiments, we applied QDFM to map gamma alpha transposons, plasmid or cosmid probes along P1 molecules, and P1 or PAC DNA clones along straightened YAC molecules ranging in size from ~490kb to >1Mbp. Our studies demonstrated the power of QDFM by showing that:
The impact of QDFM on genome research will depend on how well it scales-up to accommodate the needs of large-scale mapping and sequencing projects. Preliminary results showed that as many as 20 clones can be combed on a single microscope slide. Furthermore, QDFM is highly amenable to automation, which might increase its throughput by orders of magnitude. Molecular combing and FISH require only little user interaction and instrumentation for slide handling (washes, staining etc.) exists. Development of semi-automated slide scanning and image acquisition/analysis should facilitate these aspects of the analysis procedure. These developments should bring QDFM to the point where it is of major utility in assembly of sequence-ready physical maps and quality control during the sequencing process.
* Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research, Department of Energy, under contract DE-AC-03-76SF00098.
1 H. -U. G. Weier et al. Human Molecular Genetics 4.1903-1910 (1995).
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