Scanning force microscopy of DNA molecules elongated by convective fluid flow in an evaporating droplet.
Scanning force microscopy (SFM) was used to image intact, nearly fully elongated lambda bacteriophage DNA molecules, fixed onto freshly cleaved mica surfaces. Molecular elongation and fixation were accomplished using a newly characterized fixation technique, termed "fluid fixation." Here convective fluid flows generated within an evaporating droplet of DNA solution efficiently elongate DNA molecules for fixation onto suitably charged surfaces. SFM images of a very large bacteriophage genome, G, showed the presence of double-stranded bubbles. We speculate that these structures may contain putative replication forks. Overall, the experiments presented here demonstrate the viability of using fluid fixation for the preparation of DNA molecules for SFM imaging. The combination of largely automatable optically based techniques with the high-resolution SFM imaging presented here will likely produce a high-throughput system for detailed physical mapping of genomic DNA or clones.
Wang
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W. M. Keck Laboratory for Biomolecular Imaging, Department of Chemistry, New York University, New York, New York 10003, USA.
Lin
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J
Schwartz
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D010582:Bacteriophage lambda / Q000737:chemistry; D001435:Bacteriophages / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D004279:DNA, Viral / Q000302:isolation & purification / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008856:Microscopy, Fluorescence
D004279:DNA, Viral