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The combined AFM manipulation and fluorescence imaging of single DNA molecules
The combined AFM manipulation and fluorescence imaging of single DNA molecules
A combined fluorescence microscope/AFM set-up was constructed, which enabled the real-time manipulation of single DNA molecules. Fluorescence images of these TO-PRO-3 intercalated strands could be taken, while they were stretched with an AFM tip on silanised or polylysine covered glass surfaces. Characteristic AFM force spectra of single DNA molecules were achieved and a statistical analysis of the rupture forces, plateau heights and rupture lengths was compiled. The wide-field fluorescence images indicated that the DNA underwent condensation on polylysine to form aggregated rods and globular structures. Due to the strong unspecific adhesion of the DNA to the polylysine surface, AFM tip manipulation frequently led to strand scission. In addition, it was possible to “write” nm-sized letters of fluorescent DNA by unraveling agglomerated strands from the tip onto the surface. In contrast, DNA strands on silane showed far less condensation. Extended single chains were bound to the surface by the termini or at specific sites along the double helix. These fixed and straightened strands could be overstretched laterally to ca. 1.6 times the original contour length. Chain rupture occurred at the tip position, but occasionally mid-strand rupture was also observed. An analysis of the chain fluorescence intensity for different stretching lengths revealed that the dyes remain intercalated up to the end of the DNA B-S overstretching transition.
DNA, TO-PRO-3, atomic force spectroscopy of single molecules, real-time imaging and manipulation of single DNA molecules, TIR, total internal reflection microscopy, laser spectroscopy, single molecule, fluorescence microscopy, fluorescence imaging, AFM, TIR imaging, fluorescence filters, atomic force microscopy, DNA intercalators, DNA manipulation, cantilever tip, force spectroscopy, single DNA strands, polysaccharides, cellulose, amylose, force curves, DNA force curves, plateaus, sugar force curves, B-S transition, DNA mechanics, polymer mechanics, fluorescence dye, dye bleaching rate, DNA polymerisation, plateau force curves, rupture force, worm-like chain model, freely-jointed chain model, polylysine, silanised glass surface, DNA adhesion, hydrophobic, electrostatic, gold surface, DNA condensation, DNA nanowires, DNA rotation on silane surface, super-long DNA chains, imaging single DNA molecules, single molecule confocal microscopy, dye bleaching, antifade, slowfade, cantilever luminescence, tip fluorescence, objective-type TIR, dip-pen, water-pen, micrometer sized DNA letters, writing with DNA, single molecule manipulation, combined optical and AFM experiments, laser excitation, strand rupture, DNA sticky ends, hydrophilic, DNA knot, YOYO-1, luminescent polymer, electroluminescence, single molecule fluorescence, MEH-PPV, single molecule wire, alkyl-silanised, gas phase silanisation, silanized, octenyl-trichlorosilane, carboxymethylcellulose, DNA force spectroscopy, DNA straightening, DNA alignment, DNA concatemers, DNA rods, DNA dumbbells, dye association rate, dissociation rate kinetics, intercalator binding constant, fluorescence spectrum, evanescent field excitation, ethidium bromide, TDI, fluorophore blinking, rupture length, contour length, DNA persistence length, photobleaching, plateau forces, shoulder in force curve, enthalpic transition, strand extension and tension, polymer elasticity, chain overstretching, reversible force curve, DNA extension factor, visualisation of a single molecule rupture event.
Hards, Andrew
2004
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Hards, Andrew (2004): The combined AFM manipulation and fluorescence imaging of single DNA molecules. Dissertation, LMU München: Fakultät für Chemie und Pharmazie
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Abstract

A combined fluorescence microscope/AFM set-up was constructed, which enabled the real-time manipulation of single DNA molecules. Fluorescence images of these TO-PRO-3 intercalated strands could be taken, while they were stretched with an AFM tip on silanised or polylysine covered glass surfaces. Characteristic AFM force spectra of single DNA molecules were achieved and a statistical analysis of the rupture forces, plateau heights and rupture lengths was compiled. The wide-field fluorescence images indicated that the DNA underwent condensation on polylysine to form aggregated rods and globular structures. Due to the strong unspecific adhesion of the DNA to the polylysine surface, AFM tip manipulation frequently led to strand scission. In addition, it was possible to “write” nm-sized letters of fluorescent DNA by unraveling agglomerated strands from the tip onto the surface. In contrast, DNA strands on silane showed far less condensation. Extended single chains were bound to the surface by the termini or at specific sites along the double helix. These fixed and straightened strands could be overstretched laterally to ca. 1.6 times the original contour length. Chain rupture occurred at the tip position, but occasionally mid-strand rupture was also observed. An analysis of the chain fluorescence intensity for different stretching lengths revealed that the dyes remain intercalated up to the end of the DNA B-S overstretching transition.