DNA sequencing by synthesis using modified nucleotides and nanopore detection
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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Abstract
This disclosure is related to a method of sequencing a single-stranded DNA using deoxynucleotide polyphosphate analogues and translocation of tags from incorporated deoxynucleotide polyphosphate analogues through a nanopore.
136 Citations
40 Claims
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1. A method for sequencing a nucleic acid molecule, the method comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A compound having the structure:
Specification