We propose to use single molecule electrophoresis through nanopores to determine the base sequence of nucleic acids. The method utilizes a unique analytical system in which an electric field drives single stranded nucleic acids through the transmembrane pore of alpha-hemolysin. As the polymer traverses the pore, current is partially blocked in a manner dependent upon polymer length, concentration and composition. In practice, a single pore can transport (and potentially analyze) nucleic acid fragments at the rate of 1000 bases per second, at a cost approximately 0.1 percent of traditional methods.
The specific aims of this proposal are the following: 1. We will determine if the five naturally occurring nucleotides have blockade signatures that can be readily distinguished. 2. We will establish the shortest sequence of nucleotides that can be detected with our current prototype instrument. 3. We will determine the ability of the prototype pore to overcome secondary structure by investigating single-stranded DNA with internal hairpin loops. The overall objective of the proposed research is to demonstrate the feasibility of detecting a specific electrical signal from a single nucleotide in a strand of DNA or RNA. Our preliminary results indicate that we are approaching both the time resolution and base-specific signatures required to resolve the sequence of nucleotides in a nucleic acid strand.
|Akeson, M; Branton, D; Kasianowicz, J J et al. (1999) Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys J 77:3227-33|