NUCLEIC ACID SEQUENCING BY NANOPORE DETECTION OF TAG MOLECULES
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 individually addressable nanopores, wherein an individually addressable nanopore of said plurality of individually addressable nanopores comprises a nanopore in a membrane that is disposed adjacent to an electrode, wherein said nanopore is linked to a nucleic acid polymerase, and wherein each individually addressable nanopore is adapted to detect a tag that is released from a tagged nucleotide upon the polymerization of said tagged nucleotide;
(b) directing said nucleic acid molecule adjacent to or in proximity to said nanopore;
(c) with the aid of said polymerase, polymerizing nucleotides along said nucleic acid molecule to generate a strand that is complementary to at least a portion of said nucleic acid molecule, wherein during polymerization a tag is released from an individual nucleotide of said nucleotides, and wherein said released tag flows through or in proximity to said nanopore; and
(d) detecting the tag with the aid of said electrode, wherein the tag is detected subsequent to being released from said individual nucleotide.
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Abstract
This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.
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Citations
92 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 individually addressable nanopores, wherein an individually addressable nanopore of said plurality of individually addressable nanopores comprises a nanopore in a membrane that is disposed adjacent to an electrode, wherein said nanopore is linked to a nucleic acid polymerase, and wherein each individually addressable nanopore is adapted to detect a tag that is released from a tagged nucleotide upon the polymerization of said tagged nucleotide; (b) directing said nucleic acid molecule adjacent to or in proximity to said nanopore; (c) with the aid of said polymerase, polymerizing nucleotides along said nucleic acid molecule to generate a strand that is complementary to at least a portion of said nucleic acid molecule, wherein during polymerization a tag is released from an individual nucleotide of said nucleotides, and wherein said released tag flows through or in proximity to said nanopore; and (d) detecting the tag with the aid of said electrode, wherein the tag is detected subsequent to being released from said individual nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25)
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22. The method of claim wherein the nanopore is an alpha-hemolysin nanopore.
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26. A method for nucleic acid sequencing, the method comprising:
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a) ligating a nucleic acid hairpin onto an end of a double stranded nucleic acid molecule; b) dissociating the double stranded nucleic acid molecule and hairpin to form a single stranded nucleic acid template; c) extending a primer hybridized to the single stranded nucleic acid template using tagged nucleotides, wherein a tag associated with an individual nucleotide is released upon extension; and d) detecting the released tag with the aid of a nanopore, thereby determining the nucleic acid sequence of double stranded nucleic acid molecule. - View Dependent Claims (27, 28, 29, 30, 31)
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32. A method for nucleic acid sequencing, the method comprising:
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a) polymerizing tagged nucleotides at a first rate, wherein a tag associated with an individual nucleotide is released upon polymerization; and b) detecting the released tag by passing the tag through a nanopore at a second rate, where the second rate is greater than or equal to the first rate. - View Dependent Claims (33, 34, 35)
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36. A method for nucleic acid sequencing, the method comprising:
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a) polymerizing tagged nucleotides, wherein a tag associated with an individual nucleotide is released upon polymerization; and b) detecting the released tag with the aid of a nanopore. - View Dependent Claims (37, 38, 39, 40)
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- 41. A method for nucleic acid sequencing, comprising detecting, with the aid of a nanopore, the incorporation of a nucleotide into a nucleic acid molecule, wherein the nucleic acid molecule does not pass through the nanopore.
- 46. A method for nucleic acid sequencing, comprising detecting a byproduct of an individual nucleotide incorporation event with the aid of a nanopore.
- 50. A method for sequencing a nucleic acid molecule, comprising distinguishing between individual nucleotide incorporation events with an accuracy of greater than 4 σ
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59. A method for nucleic acid sequencing, the method comprising:
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(a) providing an array of nanopores, wherein an individual nanopore in said array is coupled to a nucleic acid polymerase; and (b) polymerizing tagged nucleotides with the polymerase, wherein an individual tagged nucleotide comprises a tag, and wherein the tag is released and detected with the aid of the nanopore. - View Dependent Claims (60, 61, 62)
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- 63. A tagged nucleotide, wherein the nucleotide comprises a tag capable of being cleaved in a nucleotide polymerization event and detected with the aid of a nanopore in a chip comprising an array of nanopores.
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67. A system for sequencing a nucleic acid molecule, comprising:
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(a) a chip comprising a plurality of individually addressable nanopores, wherein an individually addressable nanopore of said plurality of individually addressable nanopores comprises at least one nanopore in a membrane disposed adjacent to an electrode, wherein each individually addressable nanopore is adapted to aid in the detection of a tag released from a tagged nucleotide upon the incorporation of said tagged nucleotide in a nucleic acid strand that is complementary to said nucleic acid molecule; and (b) a computer processor coupled to said individually addressable nanopores, wherein said computer processor is programmed to aid in characterizing a nucleic acid sequence of said nucleic acid molecule based upon electrical signals received from said plurality of individually addressable nanopores, wherein an individual electrical signal is associated with a tag that is released from a tagged nucleotide subsequent to the incorporation of said tagged nucleotide in a nucleic acid strand that is complementary to said nucleic acid molecule. - View Dependent Claims (68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87)
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- 88. A method for sequencing a nucleic acid molecule, the method comprising providing an array of individually addressable sites at a density of at least about 500 sites per mm2, each site having a nanopore attached to a nucleic acid polymerase, and, at a given site of the array, polymerizing tagged nucleotides with a polymerase, wherein upon polymerization a tag is released and detected by a nanopore at the given site.
Specification