Nucleic acid sequencing by nanopore detection of tag molecules

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.

What is claimed is: 
     
         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.   
     
     
         2 . The method of  claim 1 , wherein said detecting of (d) further comprises identifying said tag. 
     
     
         3 . The method of  claim 2 , further comprising correlating said identified tag with a type of said individual nucleotide. 
     
     
         4 . The method of  claim 1 , further comprising generating, with the aid of a computer processor, a nucleic acid sequence of the nucleic acid molecule based upon an assessment of the tags detected during polymerization. 
     
     
         5 . The method of  claim 4 , wherein said computer processor is in a workstation that is in proximity to said chip. 
     
     
         6 . The method of  claim 1 , wherein the tag passes through the nanopore. 
     
     
         7 . The method of  claim 1 , wherein the tag passes adjacent to the nanopore. 
     
     
         8 . The method of  claim 1 , wherein said electrode is adapted to supply an electrical stimulus across said membrane. 
     
     
         9 . The method of  claim 1 , wherein said membrane has a capacitance greater than about 5 fF/μm 2  as measured across said membrane. 
     
     
         10 . The method of  claim 1 , wherein said membrane has a resistance greater than or equal to about 500 MΩ as measured across said membrane. 
     
     
         11 . The method of  claim 1 , wherein said membrane has a resistance less than or equal to about 1 GΩ across said membrane. 
     
     
         12 . The method of  claim 11 , wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 
     
     
         13 . The method of  claim 12 , wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 
     
     
         14 . The method of  claim 1 , wherein each individually addressable nanopore is adapted to regulate molecular flow. 
     
     
         15 . The method of  claim 1 , wherein said individually addressable nanopore is adapted to detect said tag upon molecular flow of said tag thereof through or adjacent to said nanopore. 
     
     
         16 . The method of  claim 1 , wherein said electrode is individually addressable. 
     
     
         17 . The method of  claim 1 , wherein said electrode is coupled to an integrated circuit that processes a signal detected with the aid of said electrode. 
     
     
         18 . The method of  claim 17 , wherein said integrated circuit comprises a logic controller. 
     
     
         19 . The method of  claim 1 , wherein said electrode is part of an integrated circuit that processes a signal detected with the aid of said electrode. 
     
     
         20 . The method of  claim 1 , wherein said membrane is a lipid bilayer. 
     
     
         21 . The method of  claim 1 , wherein the membrane is a diphytanoylphosphatidylcholine (DPhPC) lipid bilayer. 
     
     
         22 . The method of claim wherein the nanopore is an alpha-hemolysin nanopore. 
     
     
         23 . The method of  claim 1 , wherein said membrane exhibits (i) a capacitance greater than about 5 fF/μm 2  or a resistance less than or equal to about 1 GΩ across said membrane, or (ii) a capacitance greater than about 5 fF/μm 2  and a resistance less than or equal to about 1 GΩ across said membrane. 
     
     
         24 . The method of  claim 1 , wherein said membrane is disposed adjacent to a membrane compatible surface. 
     
     
         25 . The method of  claim 1 , wherein said plurality of individually addressable nanopores are at a density of at least about 500 individually addressable nanopores per mm 2 . 
     
     
         26 . A method for nucleic acid sequencing, the method comprising:
 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.   
     
     
         27 . The method of  claim 26 , further comprising directing the tag released from the individual nucleotide through the nanopore. 
     
     
         28 . The method of  claim 26 , further comprising directing the tag released from the individual nucleotide to a location adjacent to the nanopore. 
     
     
         29 . The method of  claim 26 , wherein each type of nucleotide comprises a unique tag. 
     
     
         30 . The method of  claim 26 , wherein the tag is initially attached to the 5′-phosphate of the individual nucleotide. 
     
     
         31 . The method of  claim 26 , wherein the primer is annealed to a specific position on the single stranded nucleic acid template in step (c). 
     
     
         32 . A method for nucleic acid sequencing, the method comprising:
 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.   
     
     
         33 . The method of  claim 32 , wherein the second rate is greater than the first rate. 
     
     
         34 . The method of  claim 32 , wherein each type of nucleotide comprises a unique tag. 
     
     
         35 . The method of  claim 32 , wherein the tag is initially attached to the 5′-phosphate of the individual nucleotide. 
     
     
         36 . A method for nucleic acid sequencing, the method comprising:
 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.   
     
     
         37 . The method of  claim 36 , further comprising directing the tag released from the individual nucleotide through the nanopore. 
     
     
         38 . The method of  claim 36 , further comprising directing the tag released from the individual nucleotide to a location adjacent to the nanopore. 
     
     
         39 . The method of  claim 36 , wherein each type of nucleotide comprises a unique tag. 
     
     
         40 . The method of  claim 36 , wherein the tag is initially attached to the 5′-phosphate of the individual nucleotide. 
     
     
         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. 
     
     
         42 . The method of  claim 41 , wherein tags associated with the nucleotides are released upon incorporation, and wherein subsequent to being released the tags pass through the nanopore. 
     
     
         43 . The method of  claim 41 , wherein nucleotide incorporation events are detected with an accuracy of at least 4 σ. 
     
     
         44 . The method of  claim 41 , wherein nucleotide incorporation events are detected with an accuracy of at least 5 σ. 
     
     
         45 . The method of  claim 41 , wherein nucleotide incorporation events are detected with an accuracy of at least 6 σ. 
     
     
         46 . A method for nucleic acid sequencing, comprising detecting a byproduct of an individual nucleotide incorporation event with the aid of a nanopore. 
     
     
         47 . The method of  claim 46 , wherein the nucleotide is not directly detected by said nanopore. 
     
     
         48 . The method of  claim 46 , wherein the byproduct of the nucleotide incorporation event is a tag molecule that is released upon said individual nucleotide incorporation event. 
     
     
         49 . The method of  claim 48 , wherein the tag molecule passes through the 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. 
     
     
         51 . The method of  claim 50 , wherein the accuracy is greater than 5 σ. 
     
     
         52 . The method of  claim 50 , wherein the accuracy is greater than 6 σ. 
     
     
         53 . The method of  claim 50 , wherein the nucleotide incorporation events are detected with aid of a nanopore. 
     
     
         54 . The method of  claim 53 , wherein said nanopore is an individually addressable nanopore. 
     
     
         55 . The method of  claim 53 , wherein said nanopore is in a membrane that is disposed adjacent to an electrode. 
     
     
         56 . The method of  claim 55 , wherein said electrode is in an array of electrodes at a density of at least about 500 electrodes per mm 2 . 
     
     
         57 . The method of  claim 55 , wherein said individual nucleotide incorporation events comprise the incorporation of a nucleotide in a nucleic acid strand that is complementary to said nucleic acid molecule, wherein said nucleotide comprises a tag that is released upon the incorporation of said nucleotide in said nucleic acid strand, and wherein said tag passes through or adjacent to said nanopore subsequent to being released from said nucleotide. 
     
     
         58 . The method of  claim 57 , wherein said tag is detected with the aid of said electrode subsequent to being released from said nucleotide. 
     
     
         59 . A method for nucleic acid sequencing, the method comprising:
 (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.   
     
     
         60 . The method of  claim 59 , wherein the tag passes adjacent to the nanopore subsequent to being released. 
     
     
         61 . The method of  claim 59 , wherein the tag passes through the nanopore subsequent to being released. 
     
     
         62 . The method of  claim 59 , wherein the nanopores are individually addressable. 
     
     
         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. 
     
     
         64 . The tagged nucleotide of  claim 63 , wherein the tag is attached to the 5′-phosphate of the nucleotide. 
     
     
         65 . The tagged nucleotide of  claim 63 , wherein the tag is not a fluorophore. 
     
     
         66 . The tagged nucleotide of  claim 63 , wherein the tag is detectable by its charge, shape, size, or any combination thereof. 
     
     
         67 . A system for sequencing a nucleic acid molecule, comprising:
 (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.   
     
     
         68 . The system of  claim 67 , wherein said electrode is adapted to supply an electrical stimulus across said membrane. 
     
     
         69 . The system of  claim 67 , wherein said membrane has a capacitance greater than about 5 fF/μm 2  as measured across said membrane. 
     
     
         70 . The system of  claim 67  or  69 , wherein said membrane has a resistance greater than or equal to about 500 MΩ as measured across said membrane. 
     
     
         71 . The system of  claim 67 ,  69  or  70 , wherein said membrane has a resistance less than or equal to about 1 GΩ across said membrane. 
     
     
         72 . The system of  claim 70  or  71 , wherein said resistance is as measured by opposing electrodes disposed adjacent to said membrane. 
     
     
         73 . The system of  claim 67 , wherein each individually addressable nanopore is adapted to regulate molecular flow. 
     
     
         74 . The system of  claim 73 , wherein each individually addressable nanopore is adapted to regulate molecular flow with the aid of an electrical stimulus applied to said nanopore. 
     
     
         75 . The system of  claim 67 , wherein said computer processor is in a workstation that is in proximity to said chip. 
     
     
         76 . The system of  claim 67 , wherein said computer processor is comprised in said chip. 
     
     
         77 . The system of  claim 67 , wherein said individually addressable nanopore is adapted to detect said tag upon molecular flow of said tag thereof through or adjacent to said nanopore. 
     
     
         78 . The system of  claim 67 , wherein said electrode is individually addressable. 
     
     
         79 . The system of  claim 67 , wherein said electrode is coupled to an integrated circuit that processes a signal detected with the aid of said electrode. 
     
     
         80 . The system of  claim 79 , wherein said integrated circuit comprises a logic controller. 
     
     
         81 . The system of  claim 67 , wherein said electrode is part of an integrated circuit that processes a signal detected with the aid of said electrode. 
     
     
         82 . The system of  claim 67 , wherein said membrane is a lipid bilayer. 
     
     
         83 . The system of  claim 67 , wherein the nanopore is an alpha-hemolysin nanopore. 
     
     
         84 . The system of  claim 67 , wherein the membrane is a diphytanoylphosphatidylcholine (DPhPC) lipid bilayer. 
     
     
         85 . The system of  claim 67 , wherein said membrane is disposed adjacent to a membrane compatible surface. 
     
     
         86 . The system of  claim 67 , wherein said plurality of individually addressable nanopores are at a density of at least about 500 individually addressable nanopores per mm 2 . 
     
     
         87 . The system of  claim 86 , wherein said density is at least about 1000 individually addressable nanopores per mm 2 . 
     
     
         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 mm 2 , 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. 
     
     
         89 . The method of  claim 88 , further comprising generating, with the aid of a processor, a nucleic acid sequence of the nucleic acid molecule based upon the detected tags. 
     
     
         90 . The method of  claim 88 , wherein the tag passes through the nanopore. 
     
     
         91 . The method of  claim 88 , wherein the tag passes adjacent to the nanopore. 
     
     
         92 . The method of  claims 90  or  91 , wherein the rate of polymerization is less than the rate of tag passage through or adjacent to the nanopore.