Nanopore-based resistive pulse sensing represents an important class of single-molecule measurements. It provides information about many molecules of interest (i.e. DNA, proteins, peptides, clusters, polymers, etc.) without the need for labeling. Two experiments that are especially well suited for studying with nanopore sensors are DNA sequencing and DNA-protein force measurements. This thesis will describe progress that has been made in both areas.
DNA sequencing has become an active area of research for stochastic single-molecule sensing, with many researchers striving for the ultimate goal of single-molecule de novo DNA sequencing. One intriguing method towards that goal involves the use of a DNA exonuclease or polymerase enzyme, which when attached close to the mouth of a pore, leads to cleavage of individual DNA nucleotide bases for loading into the pore for sensing. Though this method seems promising, the end goal has been elusive because the nucleotide motion is dominated by diffusion over the relevant length scales. This limits the likelihood of the cleaved nucleotide entering the pore to be characterized. The first part of this thesis will describe a method for addressing this problem, where it is shown that increasing the nucleotide capture probability can be achieved by lowering the bulk diffusion coefficient relative to the pore diffusion coefficient.
The second part of this thesis will describe the design and implementation of a new type of sensor that combines a biological nanopore experimental apparatus with optical tweezers. The goal of this apparatus is to develop a means to independently measure the force on a charged molecule inside of the pore. The setup will be thoroughly described, and preliminary results showing that it is possible to optically trap a micron sized bead within a few microns of an isolated biological nanopore while simultaneously making current measurements through that pore will be presented. This will enable force measurements on DNA molecules tethered to the bead, which opens the door for the study of molecular force interactions between DNA and biological nanopores, DNA-bound protein interactions that cause diseased states, and controlled translocation of DNA through biological nanopores.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-4889 |
| Date | 01 January 2015 |
| Creators | Brady, Kyle T |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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