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DNA sequencing by synthesis using modified nucleotides and nanopore detection

  • US 10,443,096 B2
  • Filed: 12/16/2011
  • Issued: 10/15/2019
  • Est. Priority Date: 12/17/2010
  • Status: Active Grant
First Claim
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1. A method for sequencing a nucleic acid molecule, the method comprising:

  • a) providing a chip comprising a plurality of nanopores, wherein said plurality of nanopores comprises a nanopore in a membrane adjacent to an electrode, wherein said nanopore is linked to a nucleic acid polymerase;

    b) providing at least one tagged nucleotide polyphosphate (NPP) analogue, wherein the tagged NPP comprises a tag capable of being released during polymerization catalyzed by said nucleic acid polymerase and detected with the aid of said nanopore, and wherein said tag comprises an oligonucleotide;

    c) contacting said at least one tagged NPP and the nucleic acid molecule with said nucleic acid polymerase linked to said nanopore, whereby said polymerase polymerizes said tagged NPP into a strand that is complementary to at least a portion of said nucleic acid molecule, wherein during polymerization said tag is released from said tagged nucleotide, and wherein said released tag flows through said nanopore;

    d) detecting the released tag with the aid of said electrode, wherein the tag is detected subsequent to being released from said tagged nucleotide; and

    e) correlating said tag with a type of tagged NPP, and generating, a nucleic acid sequence of the nucleic acid molecule based upon said detected tag.

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