| Summary: | 碩士 === 國立中山大學 === 化學系研究所 === 94 === The image resolution of atomic force microscopy (AFM) is still less superior to that of the electron microscopy (EM). However AFM operated in liquids complemented by Tapping-mode (TM) detection proves to be more suitable for imaging biomolecules in physiological-like environments. Nevertheless, manipulation of AFM in solution turned out to be non-trivial, several technical difficulties were encountered. In the thesis, I report using divalent cation-containing buffer as a feasible method to immobilize DNA molecules effectively for imaging in liquid media. AFM operating conditions, such as cantilever oscillating drive frequency, setpoint amplitude, feedback control parameters and scan rates were studied to obtain the optimized function. Various AFM images of Ssp I-linearized pUC19 DNA/EcoR I restriction enzyme complexes were captured, revealing the molecular details of their complex machineries. For example, the intermediate stage of the enzyme cleavage action was displayed by images showing that DNA was bent by an acute angle at the active site in the presence of one single EcoR I molecule. Some evidence for a jumping, sliding or intersegmental transfer mechanism is achieved. To trace the enzyme-DNA interaction dynamic in real time, preliminary results were obtained, but further improvements are required.
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