Nanopore based molecular detection and sequencing
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 portion of a molecule into the nanopore;
(c) varying a voltage applied across the nanopore and/or across the membrane, wherein the voltage is varied across a range of voltages from 120 mV to 150 mV;
(d) measuring the current at a plurality of applied voltages while the portion of the molecule is inserted within the nanopore; and
(e) identifying the portion of the molecule that is inserted within the nanopore based on the measured current at the plurality of applied voltages.
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 comprises a nucleic acid.
4. The method of claim 2 , 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.
5. 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.
6. The method of claim 1 , wherein the molecule is bound to a nucleotide.
7. The method of claim 6 , wherein the molecule or portion thereof is identified while the nucleotide is being incorporated into a growing nucleic acid chain.
8. The method of claim 6 , wherein the molecule is released from the nucleotide upon incorporation of the nucleotide into a growing nucleic acid chain.
9. The method of claim 8 , wherein the molecules are identified in the order in which the nucleotides are incorporated into the growing nucleic acid chain.
10. 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.
11. The method of claim 1 , wherein the current is varied according to a voltage waveform.
12. The method of claim 11 , wherein the voltage waveform is a square wave, a sinusoidal wave, a triangular wave, a saw-tooth wave, or an irregular wave.
13. The method of claim 1 , wherein said voltage is varied by applying an alternating current waveform to said nanopore and/or membrane.
14. The method of claim 13 , wherein said alternating current waveform has a frequency of about 100 Hz.