Formation and Characterization of DNA Concatemers for Single Molecule Mechanism Studies
Well known as genetic information carrier, DNA however, in the past three decade experienced a revolution to become a fashionable fabrication material in nanotechnology as a consequence of the high predictability and accuracy of base-paring recognition. Although DNA can be constructed in many fancy 2D and 3D structures, this thesis work focused on constructing one-dimension linear DNA concatemers in order to study sequence specific DNA-ligand binding at the single-molecule level.
This thesis presents two methods of assembly of DNA concatemers by synthetic oligonucleotides. The first method investigated hybridization of desired oligonucleotides in
solution with aiming to optimize the length of the concatemers. Gel electrophoresis and AFM were used to check the size and shapes of the formed concatemers. Results showed by inceasing ionic strength at 200 mM NaCl, concatemers were formed most quickly. Concatemers with lengths up to several hundred base pairs was successfully constructed and separated from those non-linear shaped DNA.
With the same purpose of forming linear DNA concatemers, the second method provide a strategy of building DNA concatemers on a solid surface with the same sequence of oligonucleotides. Biotin-oligonucleotides were involved to immobilize on the surface. Furthermore, to avoid formation of non-linear DNA constructs, DNA were hybridized layer by layer. QCM-D was used to monitor the process of construction as well as study the viscoelasticity of the formed DNA layers. Result indicated that DNA multilayers were constructed. To confirm this, constructed DNA were cleaved off the surface and checked for sizes by running in gel electrophoresis. Clear bands in the image strongly supported that each hybridization step on surface was complete and DNA concatemers were successfully constructed.