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Compositions, devices, systems, for using a Nanopore

  • US 20100035260A1
  • Filed: 06/26/2009
  • Published: 02/11/2010
  • Est. Priority Date: 04/04/2007
  • Status: Active Grant
First Claim
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1. A method for controlling movement of a polynucleotide using voltage feedback control, the method resulting in identifying the sequence of a polynucleotide, the method comprising the steps of:

  • providing two separate adjacent chambers comprising a medium, an interface between the two chambers, the interface comprising a material having at least channel therethrough and wherein one chamber is on the cis-side of the interface and the other chamber is on the trans-side of the interface, the channel so dimensioned as to allow sequential monomer-by-monomer passage from the cis-side of the channel to the trans-side of the channel of only one polynucleotide strand at a time;

    providing an enzyme having binding activity for a polynucleotide;

    providing a blocking oligomer;

    providing a polynucleotide complex, wherein a portion of the polynucleotide complex is double-stranded and a portion is single-stranded;

    providing a complimentary oligomer, wherein the complimentary oligomer is complementary to a portion of the single stranded polynucleotide;

    providing a substrate;

    introducing the polynucleotide complex into one of the two chambers;

    introducing the blocking oligomer into the same chamber;

    allowing the blocking oligomer to bind to the polynucleotide complex;

    introducing the enzyme into the same chamber;

    introducing the complementary oligomer into the other chamber;

    applying a potential difference between the two chambers, thereby creating a first polarity, the first polarity causing the single stranded portion of the polynucleotide to transpose through the channel to the trans-side thereby stripping the blocking oligomer from the polynucleotide complex;

    measuring the electrical current through the channel thereby detecting the polynucleotide;

    decreasing the potential difference a first time, thereby creating a second polarity;

    allowing the complementary oligomer to bind to the single-stranded polynucleotide;

    reversing the potential difference, thereby creating a third polarity;

    providing conditions to allow the enzyme to bind to the polynucleotide complex;

    providing conditions to allow the enzyme to incorporate substrate into the polynucleotide, thereby increasing length of the double-stranded portion;

    reversing the potential difference a second time;

    measuring the electrical current through the channel, thereby detecting a polynucleotide having incorporated substrate or a polynucleotide bound to the enzyme;

    repeating any one of the steps, thereby controlling the synthesis of the polynucleotide.

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