Ma, Ziyang (2008) Precision optical measurements of DNA structure and synthesis. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-11212007-084606
The structure and synthesis of DNA is important. Traditionally,high-resolution structures are solved with X-ray crystallography, and kinetic measurements are taken with consumption of large amount of reagents. In this thesis, I developed new optical methods to probe the DNA structure and to measure kinetics of DNA synthesis. I measured the helical rise of A-form DNA at the single molecule level using a near-field optical microscope that has sub-10 nm resolution. The microscope is a combination of an atomic force microscope (AFM) and a fluorescence microscope. Imaging at the single fluorophore level was allowed with the development of a novel phase filter that gives $sim$ 6 fold improvement in the signal-to-noise ratio. I was able to reliably and repeatedly image pairs of molecules separated by ~ 15 nm and demonstrate the first true Rayleigh resolution test for near-field images. Various evidences, such as crystal structures and sequencing experiments, indicate that the primer-template DNA sequence may affect nucleotide incorporation. I developed a droplet-based microfluidic fast-mixing device that requires minimal sample consumption and measured kinetics of nucleotide incorporation with DNA sequence permutation. I showed that with careful and systematic kinetic measurements, one can obtain rich information of the process of DNA synthesis.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||DNA; DNA polymerase; droplet; fast mixing; kinetics; microfluidics; near-field optical microscopy|
|Degree Grantor:||California Institute of Technology|
|Division:||Physics, Mathematics and Astronomy|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||6 September 2007|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||11 Dec 2007|
|Last Modified:||26 Dec 2012 03:10|
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