PROJECT SUMMARY We seek a new technique for very high speed characterization and sequencing of polynucleotides such as DNA. The approach relies on the notion that a linear polymer passing through a suitably small channel or pore in an otherwise impermeable interface will sequentially modify the local properties of the pore in a manner that reflects the sequence of monomers in the polymer. Our recent results using model RNA and DNA polymers that were electrophoretically driven through a protein channel in a lipid bilayer demonstrate the promise of this approach, but are limited by the delicacy of the organic protein-lipid interface which cannot tolerate the denaturants and temperatures required to eliminate base pairing and other secondary structures in native polynucleotides. The short term goals of this proposal are to develop a robust, inorganic interface (silicon nitride) that will duplicate the results obtained with the organic interface and lead to the development of an instrument for very high speed sizing of native polynucleotides. Accomplishing these short term goals will demonstrate the feasibility of using a small channel in an inorganic interface as the base on which to develop a novel sensing device for high speed sequencing of single DNA molecules. Because this approach eliminates several of the currently required preparatory chemical steps in DNA sequencing, and because it depends on inherently rapid, molecular events, success with this method will be an extraordinarily important advance that should reduce the time and cost of nucleic acid sequencing by several orders of magnitude.
