A solid-state nanopore-based platform for molecular diagnostics
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The ability to identify and characterize infectious pathogens is essential for proper diagnosis, treatment and disease management. Many pathogens display similar pathophysiological traits, but respond to different treatment regimes and can develop resistance to prescribed treatments. This creates a need for development of novel methods to provide accurate, rapid, and cost-effective genomic characterization of infecting pathogens. While diagnostic methods that utilize a pathogen's genomic information do exist today, currently their use is severely hindered by high costs, methodological complexity, and lengthy turnaround. One relatively new approach that could address these issues utilizes the known genomic sequence variations of pathogen strains to create a barcode of detectable genomic 'tags', specific to each strain. Unfortunately, current optical methods to detect such 'tags' lack accuracy and resolution. This project explores the feasibility of using solid-state nanopores to circumvent these apparent roadblocks, laying the foundation for a purely electronic pathogen barcoding method. The first aim of this project is to evaluate the platform's ability to detect minute fluctuations in the DNA's helical structure through analysis of transient changes to the ion current as the DNA passes through a nanopore. The second aim of this project is to detect local points of DNA structure variation. In this aim we utilize highly sequence-specific synthetic peptide nucleic acid (PNA) probes to locally tag predesigned short sequences along a DNA molecule. The sequence specificity of the tags and the nanopore's ability to discriminate between tagged and untagged regions along the DNA allow identification of specific sequences in a long DNA molecule. The final aim of this project is to develop the ability to localize the PNA probes along the DNA molecule. This enables us to conduct a proof-of-concept demonstration of the combined PNAjnanopore method through discrimination between two variants of a gene from two nearly identical human immunodeficiency virus (HIV) subtypes. This body of work forms the foundation for a highly capable nanopore-based molecular diagnostics platform.
Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at firstname.lastname@example.org. Thank you.