UBC Theses and Dissertations
Detection and analysis of nucleic acids using nanometer-scale pores Nakane, Jonathan Jamie
Advances in genomics and in its application to personalized medicine have increased the demand for faster and cheaper methods of nucleic acid analysis techniques. Nanopore-based single-molecule detection schemes are exceptionally good candidates for novel technologies to address this demand. This thesis presents the initial attempts and characterization of two implementations of the alpha-hemolysin (aHL) protein nanopore as a nanosensor for the detection and analysis of single nucleic acid molecules. The two detection schemes address shortcomings in present analysis techniques, and are distinguished by the mode of molecule capture and identification and by the proposed application. In the first method, single-stranded DNA fragments are captured from the bulk solution on the cis -side of the pore membrane, with the resulting rate of capture used as an electronic means of measuring local nucleic acid concentrations, obviating the need for optics or fluorescent molecular tags used during conventional DNA detection. Experimental results of single-stranded DNA fragment capture at high transmembrane potentials and an improved model of molecule capture from the bulk solution show that nanopore capture rates may be a viable method of estimating nucleic acid concentrations. In the second method, the aHL pore is used in conjunction with a synthetic probe molecule to capture individual single stranded DNA molecules on the trans -side of the membrane to perform single-molecule hybridization and force-dissociation experiments. Experimental results demonstrate that ensemble measurements can be used to measure the kinetics of hybridization and dissociation of single molecules of nucleic acids hybridizing to the probe molecule, with single-molecule sensitivity and single-nucleotide specificity. The detection of molecules across a membrane using force-dissociation techniques shows potential in use for the next generation of instrumentation for genomics and potentially for in vivo, real-time detection of biomolecules.
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