There is a great need to reduce the cost of DNA sequencing to achieve the goal of the $1000 genome. We recently developed a new nanopore-based sequencing by synthesis (Nano-SBS) approach. In this project, we will pursue the development of the Nano-SBS approach into a high throughput real-time single-molecule sequencing platform. In the Nano-SBS method, a polymer tag of distinct size and charge is attached to the terminal phosphate of each of the four nucleotides. When the complementary nucleotide analog enters a template-primer-polymerase complex that is attached to the nanopore during the polymerase reaction, the tag specific for that nucleotide is captured in the voltage gradient within the nanopore and results in a current blockade unique to each tag for sequence determination. The polymerase is covalently attached to the nanopore by a short linker so the polymeric tag will have sufficient time to enter the vestibule and constriction of the nanopore prior to its release ensuring that its current blockade signal is recorded by the nanopore. The extended DNA strand bears only natural nucleotides, enabling long reads. We have carried out the key proof-of-principle experiments to demonstrate the feasibility of this approach. Here our strong team of nucleic acid chemists, genomic scientists, electrical engineers, and nanofabrication experts will further develop the Nano-SBS as a high throughput genomic sequencing system. We will develop robust methodology to attach polymerase to the .-hemolysin (AHL) nanopore and synthesize nano-tags with unique chemical properties resulting in AHL current blockades distinct from each other and nucleotide precursors. We will test these elements in single pores as well as in new nanopore array chips with separate sensors and circuits for each pore. We will produce mutant AHL and polymerase constructs and link them to each other, selecting for the combination that assures accurate DNA extension reactions, and rapid capture and detection of tags in nanopores. The nanopore chips will be enhanced and expanded from the current 260 nanopores to over 125,000 using advanced nanofabrication techniques. We will conduct real-time single molecule Nano-SBS on DNA templates with known sequences to test and optimize the overall system. These research and development efforts will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine. 1
There is a great need to reduce the cost of DNA sequencing to achieve the goal of the $1000 genome. The proposed Nano-SBS system will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine. (NOTE: The three headings listed above must be included in the document that is submitted even if a particular section had no changes from the previous submission. If there are no changes for a section include the header but leave the text area blank to ensure appropriate processing of this information by NIH's electronic systems. ) 3
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| Fuller, Carl W; Kumar, Shiv; Porel, Mintu et al. (2016) Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array. Proc Natl Acad Sci U S A 113:5233-8 |
| Stranges, P Benjamin; Palla, Mirkó; Kalachikov, Sergey et al. (2016) Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array. Proc Natl Acad Sci U S A 113:E6749-E6756 |
| Kasianowicz, John J; Balijepalli, Arvind K; Ettedgui, Jessica et al. (2016) Analytical applications for pore-forming proteins. Biochim Biophys Acta 1858:593-606 |
| Silin, Vitalii; Kasianowicz, John J; Michelman-Ribeiro, Ariel et al. (2016) Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins. Membranes (Basel) 6: |
| Balijepalli, Arvind; Ettedgui, Jessica; Cornio, Andrew T et al. (2014) Quantifying short-lived events in multistate ionic current measurements. ACS Nano 8:1547-53 |
