UBC Theses and Dissertations
Genotyping by nanopore force spectroscopy : method development and evaluation for clinical diagnostics Wiggin, Matthew John
Clinical diagnostic genotyping has the potential to predict an individual’s response to a prescribed drug, and could thus dramatically improve drug efficacy and reduce adverse drug interactions. However, widespread implementation of clinical diagnostic genotyping is currently prevented by a lack of fast, simple clinical genotyping platforms. This thesis describes the development of a new genotyping technique based on nanopore force spectroscopy (NFS) which may fulfill this need, and serves as a feasibility study for further development towards a commercial instrument. The thesis begins by describing NFS, which is a novel, general technique used to detect bio-molecules and characterize their physical interactions with one another. NFS is applied to base-calling by forming a duplex between an engineered single-stranded DNA probe and a DNA sample, and then measuring the dissociation rate under an applied force. The dissociation rate is shown to be extremely sensitive to duplex sequence homology: tests using purified synthetic DNA fourteen bases long demonstrate that even a single base mismatch can increase the dissociation rate over 100-fold. This high specificity, combined with the sensitivity of nanopore detection, allows a base-call to be made from as few as 100 single molecule dissociation events involving the target. Based on these results, it is estimated that with further development, NFS genotyping could be possible from purified, unlabeled genomic DNA in less than 1 hour, without requiring PCR amplification. These characteristics would make NFS extremely attractive as a clinical diagnostic genotyping technology. Further development is still required to produce an instrument capable of testing genomic DNA. However, based on the success of tests so far, this thesis concludes that further development of such an instrument is clearly warranted, especially given its potential impact on human health.
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