To improve DNA sequencing and to develop practical methods of RNA sequencing, this NHGRI Phase I project focuses on using solid-state nanopore sensors coupled with sub-30nm wide channels embedded into silicon nitride, with electronics operating at 10 MHz bandwidth for DNA sequencing and direct RNA sequencing. The basic concept involves using an applied voltage to drive single-stranded DNA molecules through a narrow nanopore, which separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the nanopore they modify the flow of ions, and structural information can be extracted by analysis of the duration and magnitude of the resulting current reductions. The proposed nanochannel system solves two issues relating to DNA sequencing with solid-state nanopores: 1) feeding long strands of DNA to the sensing element in a single-stranded conformation; 2) reducing the variability found in DNA translocation signals by decreasing the conformation variance of DNA within the nanopore interior. Specifically, we seek to make solid-state ionic-current based nanopore sequencing possible by combining three important components: a nanochannel with sufficiently tight dimensions to allow long strands of DNA to enter the sensing nanopore in an ideal conformation, ultra-thin nanopores to increase signal-to-noise and reduce the number of bases within the pore interior, and optimally fast measurement of translocation through these pores with low-noise, high-bandwidth electronics. Our approach aims to eliminate the need for any enzymes and enables DNA molecules to be geometrically constrained and controlled as they are guided to the nanopores.
The proposal addresses key obstacles that must be overcome to achieve enzyme-less nanopore- based low-cost high-speed sequencing of chromosomal length DNA molecules. This project will help improve the quality and efficiency of DNA sequencing and enable direct RNA sequencing (e.g., longer read lengths, faster turn-around time, greater accuracy, and higher-throughput etc.) at reasonable costs with the anticipation that significant advances in any of these and related areas would make significant contributions to the mission of NHGRI and the field of genomics, including to many of NHGRI's other technology development goals.