D. P. Allison, P. S. Kerper, M. J. Doktycz, T. Thundat, and R. J. Warmack
Health Sciences Research Division*, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123
Physical mapping of genomic DNA at 100 base resolution using direct AFM imaging has been accomplished. This new mapping technology was created by attaching a mutant EcoRI endonuclease,[1] deficient in cleavage function, to double-stranded DNA and imaging the attachment sites at high resolution by AFM. Preliminary results of mapping plasmids ( 3-7 kb ), and lambda DNA (50 kb) by this direct imaging technique, suggest that this technology could be applied to map cosmid-sized clones at accelerated rates.
The potential for EcoRI restriction mapping larger clones, such as BAC's and YAC's is frustrated only by the molecules becoming entangled on the mounting surface. However, by integrating techniques involving anchoring one end of a DNA molecule to a surface and straightening the molecule by either shear or electrophoretic forces, these larger DNA's could also be mapped by direct AFM imaging. As a consequence existing gaps in contig maps, due to DNA fragments that resist being cloned, would be eliminated.
Improvements in commercial AFM instrumentation combined with our efforts to streamline sample preparation have made this a rapid and reliable technique. This coupled with improvements making existing software more "user friendly," by integrating image acquisition with image analysis, should make this new mapping technology readily transferable to laboratories lacking experience with scanning probe microscopes. Using preliminary results as a benchmark, this new methodology should be capable of mapping one cosmid per day.
[1] D. J. Wright, K. King, and P. Modrich, "The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease," J. Bio. Chem. 264(20), 1 1816-21, 1989.
* Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy under contract DE-AC05-840R21400 with Lockheed Martin Energy Systems, Inc.