Nanopore Based Molecular Detection and Sequencing

Abstract

This disclosure provides systems and methods for molecular identification and polymer (e.g., nucleic acid) sequencing using nanopores. The polymer may be passed through or in proximity to the nanopore and various subunits of the polymer may affect the current flowing through the nanopore. The various subunits may be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. In some cases, the polymerization of tagged nucleotides presents tag molecules to the nanopore that can be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. Also provided herein are systems and methods for sequencing both the sense and anti-sense strand of a double stranded nucleic acid molecule with a nanopore and methods for using ribonucleic acid (RNA) speed bump molecules to slow the passage of a nucleic acid molecule through or in proximity to a nanopore.

1 . A method for identifying a molecule or portion thereof, the method comprising:
 (a) providing a chip comprising at least one nanopore in a membrane that is disposed adjacent or in proximity to an electrode, wherein the electrode is adapted to detect a current passing through the nanopore;   (b) inserting a molecule or portion thereof into the nanopore;   (c) varying a voltage applied across the nanopore and/or across the membrane; and   (d) measuring the current at a plurality of voltages to identify the molecule or portion thereof.   
     
     
         2 . The method of  claim 1 , wherein the molecule is a polymer molecule and portions of the polymer molecule are identified as the polymer molecule passes through, or resides in, the nanopore. 
     
     
         3 . The method of  claim 2 , wherein the polymer molecule is a nucleic acid and the portions of the polymer molecule are nucleic acids or groups of nucleic acids. 
     
     
         4 - 6 . (canceled) 
     
     
         7 . The method of  claim 2 , wherein the polymer molecule is trapped in the nanopore. 
     
     
         8 . The method of  claim 7 , wherein the polymer molecule is threaded back and forth through the nanopore to identify at least portions of the polymer molecule a plurality of times. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 2 , wherein the portions of the polymer molecule are identified in the order in which they are along the length of the polymer molecule. 
     
     
         11 . The method of  claim 1 , wherein the molecule is bound to a nucleotide. 
     
     
         12 . The method of  claim 11 , wherein the molecule or portion thereof is identified while the nucleotide is being incorporated into a growing nucleic acid chain. 
     
     
         13 . The method of  claim 11 , wherein the molecule is released from the nucleotide upon incorporation of the nucleotide into a growing nucleic acid chain. 
     
     
         14 . The method of  claim 13 , wherein the molecules are identified in the order in which the nucleotides are incorporated into the growing nucleic acid chain. 
     
     
         15 . The method of  claim 1 , wherein the current at a plurality of voltages comprises an electronic signature and (d) further comprises comparing the electronic signature to a plurality of reference electronic signatures to identify the molecule or portion thereof. 
     
     
         16 . The method of  claim 1 , wherein the current is varied according to a voltage waveform. 
     
     
         17 . The method of  claim 16 , wherein the voltage waveform is a square wave, a sinusoidal wave, a triangular wave, a saw-tooth wave, or an irregular wave. 
     
     
         18 - 21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein said voltage is varied by applying an alternating current (AC) waveform to said nanopore and/or membrane. 
     
     
         23 . The method of  claim 22 , wherein said AC waveform has a frequency of at least about 100 Hz. 
     
     
         24 - 36 . (canceled) 
     
     
         37 . A method for sequencing a nucleic acid molecule or portion thereof, the method comprising:
 (a) passing a single stranded nucleic acid molecule through or in proximity to a nanopore in a membrane that is disposed adjacent or in proximity to an electrode, wherein said single stranded nucleic molecule comprises a sense strand coupled to an anti-sense strand through a nucleic acid segment ligated on an end portion of each of said sense strand and anti-sense strand, and wherein the electrode is adapted to detect an electrical current upon the single stranded nucleic molecule passing through or in proximity to the nanopore;   (b) obtaining, with the aid of said electrode, current measurements while passing the single stranded nucleic acid molecule through or in proximity to the nanopore; and   (c) determining a sequence of said single stranded nucleic acid molecule from current measurements obtained in (b).   
     
     
         38 . The method of  claim 37 , wherein determining said sequence of said single stranded nucleic acid molecule in (c) further comprises determining a sequence of a double stranded nucleic molecule comprising said sense strand and said anti-sense strand. 
     
     
         39 . The method of  claim 37 , wherein the nucleic acid segment is a nucleic acid hairpin. 
     
     
         40 - 43 . (canceled) 
     
     
         44 . A method for sequencing a nucleic acid molecule, comprising:
 (a) providing a chip comprising at least one nanopore in a membrane that is disposed adjacent or in proximity to an electrode, wherein the electrode is adapted to detect the nucleic acid molecule or a portion thereof;   (b) directing the nucleic acid molecule through or in proximity to the nanopore, wherein progression of the nucleic acid molecule through or in proximity to the nanopore is stopped or stalled with the aid of at least one ribonucleic acid (RNA) speed-bump molecule associated with the nucleic acid molecule; and   (c) sequencing the nucleic acid molecule or a portion thereof as the nucleic acid molecule passes through or in proximity to the nanopore.   
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 44 , wherein the RNA speed-bump molecule comprises an oligonucleotide containing a sequence of one or more oligonucleotide bases. 
     
     
         47 - 75 . (canceled)