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NANOPORE-BASED SINGLE DNA MOLECULE CHARACTERIZATION USING SPEED BUMPS

  • US 20120160688A1
  • Filed: 12/21/2011
  • Published: 06/28/2012
  • Est. Priority Date: 12/22/2010
  • Status: Active Grant
First Claim
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1. A method of obtaining sequence information of an unknown sequence in a ss test DNA, comprising:

  • (B1) forming a first bulky structure (BS1) on a first end of the ss test DNA at a first temperature,(B2) applying a first electric potential to flow the ss test DNA through a nanopore,(B3) forming a second bulky structure (BS2) on a second end of the ss test DNA at a second temperature,(B4) optionally applying another electric potential to reverse the flow of the ss test DNA until the ss test DNA is stopped by BS2 before the constriction area of the nanopore,(B5) contacting a random speed bump pool with the ss test DNA to form a speed bump-test DNA complex having at least one speed bump-test DNA duplex segment at a working temperature,(B6) applying a third electric potential to flow the speed bump-test DNA complex through the nanopore until a first speed bump-test DNA duplex segment is stopped before the constriction area of the nanopore,(B7) obtaining a first set of electrical signals when the first speed bump-test DNA duplex segment is stalled inside the nanopore for a dwelling time, and characterizing the nucleotide sequence that is in front of the first speed bump-test DNA duplex segment and the first basepair of the first speed bump-test DNA duplex segment, in the flow direction of the ss test DNA,(B8) dissociating the first speed bump-test DNA duplex segment and continuing the flow of the DNA through the nanopore,(B9) repeating steps (B4)˜

    (B8) until the ss test DNA is stopped by BS1 or BS2,(B10) applying another electric potential to move the test DNA at a reversed direction of the test DNA flow in step (B5) until the ss test DNA is stopped by the other bulky structure before the constriction area of the nanopore,(B11) repeating steps (B9)˜

    (B10) at least 1 time, and(B12) constructing the ss unknown DNA sequence by overlapping the collected nucleotide sequence information,

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