New polyacrylamide gel-based methods of sample preparation for optical microscopy: immobilization of DNA molecules for optical mapping.
New methods have been developed for rapid immobilization of biological macromolecules and other microscopic objects from aqueous solution at gel/gel, gel/solid and gel/solution interfaces using thin polyacrylamide gels covalently bound to glass surfaces. When quickly spread over a dry gel, an aqueous sample loses most of its water and low-molecular-weight solutes due to migration of these components into the gel. All optically observable objects thus become concentrated at the gel surface and may be easily located by light microscopy. Based on this, a procedure for binding DNA at a positively charged gel/solution interface was developed. A mild immobilization of the DNA molecules was obtained, allowing 'all in focus' observations of DNA digestion by restriction endonucleases with an apparent rate close to that in solution.
Morozov
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W.M. Keck Foundation Laboratory for Biomolecular Imaging, Department of Chemistry, New York University, New York 10003, USA.
Morozova
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Hiort
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Schwartz
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D000178:Acrylamides; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004271:DNA, Fungal / Q000648:ultrastructure*; D005782:Gels / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry
D000178:Acrylamides; D004271:DNA, Fungal; D005782:Gels; D004262:DNA Restriction Enzymes