Precision Optical Measurements of DNA Structure and Synthesis

Citation

Ma, Ziyang (2008) Precision Optical Measurements of DNA Structure and Synthesis. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/VKNW-BS41. https://resolver.caltech.edu/CaltechETD:etd-11212007-084606

Abstract

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 ~ 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.

Thesis Files