Title: Development of Single Molecule Electronic SNP Assays using Polymer Tagged Nucleotides and Nanopore Detection
Description: As knowledge of the human genome has accelerated, various diseases and individuals’ responses to drugs have been pinpointed to specific DNA variations in one’s genome. Among many different types of variants, the most common and simplest is the single nucleotide polymorphism (SNP) in which a single base substitution occurs. Although there have been considerable improvements in technologies that can reveal a single base difference in a DNA strand, simple and affordable methods that have high detection sensitivity and require small sample volume are expected to facilitate widespread adoption of routine SNP analysis in clinical settings. One such method that meets these requirements is to use nanopore as a single molecule detector, an emerging analytic system that detects changes in current related to molecules occupying a nanometer aperture. This dissertation thus chronicles our endeavors in developing a nanopore-based SNP assay using polymer tagged dideoxynucleotides (ddNTPs). The fundamental principles of this method rely on single base extension (SBE) of a primer by DNA polymerase using polymer tagged ddNTP analogs for allele discrimination and simple electronic readout of an alpha hemolysin (αHL) nanopore current for allele detection at the single molecule level.

Using four uniquely tagged ddNTPs, a characteristic current level that is specific to each base is produced, thus identifying the SNP alleles present and the genotype at the site. To demonstrate the feasibility of this approach, four polymer attached ddNTPs, each with a different tag that generates a characteristic current blockade level in the αHL nanopore, were designed and synthesized. To search for a DNA polymerase that can accept these tagged ddNTP analogs as substrates, several candidate DNA polymerases were surveyed and their relative efficiencies for incorporation of the analogs were compared (Chapter 2). To generate a steady and stable blockade event for accurate SNP analysis, two different means of positioning a tag molecule in the αHL nanopore after the SBE reaction have been explored: covalent conjugation of DNA primer to the pore and immobilization of biotinylated primer within the pore by streptavidin. To find a suitable position for primer attachment on the pore, three αHL mutants, each with a different single conjugation point, were constructed.

Using these mutants, different DNA-pore conjugates were produced and purified via various chromatography systems (Chapter 3). In the nanopore system, charged molecules such as DNA are electrophoretically driven through the pore under an applied voltage, thereby modulating the ionic current through the nanopore. This current reveals useful information about the structure and dynamic motion of the molecule at the single molecule level. Before performing SNP analysis, we first studied single molecule behaviors of oligonucleotides of different lengths and structures in the αHL pore and their ensuing current signatures in the system (Chapter 4). Finally, harnessed with tools and insights from the nanopore single molecule studies, actual SNP assays were performed in our nanopore system using the polymer tagged ddNTPs and SBE. Chapter 5 discusses the integrated approach where SBE is achieved on a primer-conjugated αHL nanopore and Chapter 6 presents the results using a biotin-streptavidin complex for immobilization of tag molecules in the pore.

Overall, this thesis validates adaptation of the nanopore detection system for SNP analysis using the polymer tagged ddNTPs.