| The ability to read and sequence genomic DNA is critical to developing an improved understanding of disease, disease evolution, and the design of effective drugs. Low-cost, ultra-fast DNA sequencing has the potential to revolutionize healthcare by enabling individual genomes to be fully determined through routine procedures. Although investment towards advancing sequencing technologies has resulted in significant improvements in both cost and time, further progress is needed to reach the goal of "personalized medicine". One such promising approach is nanoporebased sequencing. Our lab is developing a novel nanopore-based DNA sequencing platform based on optical detection of double-stranded DNA (dsDNA) as it is "unzipped" by solid-state nanopores. Unzipping is the rupturing of the hydrogen bonds of dsDNA, which separates the molecule into its single-stranded components. The unzipping process slows the molecule's passage through a nanopore so that it can be optically detected. Although protein nanopores have been used to extensively explore the kinetics of DNA unzipping, to date there has been no study of these kinetics using solid-state nanopores. The first aim of this project is to the determine the feasibility of using solid-state nanopores to unzip DNA. The use of solid-state nanopores in our sequencing methodology is essential, as they permit massive parallelism through the formation of nanopore arrays. We use optical detection to facilitate simultaneous parallel detection from an entire array of pores, which dramatically increases the throughput. The second aim of this work is to demonstrate optical detection of nanopore unzipping events. This is the cornerstone of the readout process and key in the proof of principle of this sequencing technology. Given the short distance between two bases in native DNA (∼0.4 nm) and the geometry of a solid-state nanopore, a DNA expansion step is needed to enable differentiation among the bases. The final aim of this study is to develop a biochemical procedure to achieve this expansion, whereby each base in a given DNA strand is converted into a longer, predefined sequence. This total body of work forms the foundation and proof of concept for a highly promising nanopore sequencing methodology. |
