Introduction to the Workshop
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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
High Resolution Mapping by Scanning Probe Microscopy
D. P. Allison, T. Thundat, and R. J. Warmack
Health Sciences Research Division, Oak Ridge National Laboratory and
Colin Collins* and Joe Gray*#
*Lawrence Berkeley National Laboratory, Resource for Molecular Cytogenetics, #University of California San Francisco, Division of Molecular Cytometry
The aim of this project is to evaluate the feasibility of using atomic force microscopy (AFM) to image protein specifically bound to large DNA molecules and to apply this technology to the rapid construction of "restriction mapped" long range P1, BAC and PAC contigs. Substantial progress toward this goal has been accomplished.
Recent improvements in our techniques for mounting DNA to pristine mica surfaces and the incorporation of critical point drying into our methodology have allowed us to image DNA relatively free from background noise. We have imaged large molecules, such as 50 kb lambda DNA and 80 kb P1 clones, and anticipate no problems imaging molecules up to 200 kb. Restriction mapping will be accomplished by identifying site specific binding of restriction enzymes that have been modified to bind but not cleave DNA. Model experiments using such a mutant EcoRI restriction enzyme have identified the single EcoRI site on pBS+ and the two EcoRI sites on pMP32 plasmid DNA. Visualization of the restriction sites is greatly facilitated by biotinylating the enzyme-DNA complex and labeling with 10nm streptavidin-gold prior to image acquisition. In an extension of this technology we are attempting to map oligonucleotides to cloned DNA by RecA protein mediated hybridization and imaging RecA attachment sites or D-loop sites.
This technology offers the possibility to radically accelerate the construction of long range P1, PAC and BAC contigs for use in large scale directed sequencing and positional cloning projects. A single technician using a pair of AFM microscopes ($80,000) can be expected to image and assemble 50 Mb per year. Since AFM can potentially image both protein (transcription factors) and small oligonucleotides (corresponding to exons, STSs, and promoter elements) specifically bound to DNA, it may become possible to rapidly construct fully integrated physical and genetic maps for any chromosomal region.