24 June 1993 Atomic force microscopy of DNA-colloidal gold and DNA-protein complexes
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Proceedings Volume 1891, Advances in DNA Sequencing Technology; (1993) https://doi.org/10.1117/12.146706
Event: OE/LASE'93: Optics, Electro-Optics, and Laser Applications in Scienceand Engineering, 1993, Los Angeles, CA, United States
Abstract
We are developing methods for random and site-specific labeling of individual DNA molecules to facilitate manipulation of fragments excised in the atomic force microscope (AFM) and for localization of specific DNA domains, such as protein binding sites and origins of replication. One successful method was to incorporate biotinylated nucleotides at random internal locations or specifically at the ends of linearized DNA molecules in vitro. Following complex formation with 5 nm diameter streptavidin-gold conjugates, chromatographic purification and passive adsorption of the complexes of mica, the biotinylated domains were easily localized in the AFM by virtue of the distinctive size and shape of the streptavidin-gold complex. In many cases unconjugated streptavidin (i.e., lacking gold) was also observed attached to the biotinylated DNA. A second approach to site-specific labeling of DNA for imaging in the AFM was to react DNA with restriction enzymes having sequence-specific binding properties. Like the unconjugated streptavidin-DNA complexes, these enzyme-DNA complexes were visible without attached colloidal gold. Efforts to image DNA labeled in vivo using bromodeoxyuridine (BrdU) and anti-BrdU antibodies are ongoing.
© (1993) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Luming Niu, Wenling Shaiu, James Vesenka, Drena D. Larson, Eric Henderson, "Atomic force microscopy of DNA-colloidal gold and DNA-protein complexes", Proc. SPIE 1891, Advances in DNA Sequencing Technology, (24 June 1993); doi: 10.1117/12.146706; https://doi.org/10.1117/12.146706
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