Method and device for identifying nucleotide, and method and device for determining nucleotide sequence of polynucleotide

• US 10,202,644 B2
• Filed: 10/14/2015
• Issued: 02/12/2019
• Est. Priority Date: 03/03/2010
• Status: Active Grant

• Current Assignee: Osaka University
• Original Assignee: Quantum Biosystems Inc.
• Inventors: Taniguchi, Masateru, Tsutsui, Makusu, Yokota, Kazumichi, Kawai, Tomoji
• Primary Examiner(s): Ball, J. Christopher
• Application Number:
  US14/883,494
• Publication Number:
  US 20160138101A1
• Time in Patent Office: 1,217 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/157 Nanotubes or nanorods
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

The present invention provides technology that uses current measurements to identify nucleotides and determine a nucleotide sequence in polynucleotides. The present invention calculates a modal value of a tunnel current that arises when a nucleotide or polynucleotide for analysis passes through between electrodes, and then employs the calculated modal value. The present invention accordingly enables direct rapid implementation to identify nucleotides and to determine a nucleotide sequence in a polynucleotide without marking.

Claims

1. A method for sequencing a polynucleotide, comprising:

(a) providing a solid substrate comprising two electrodes that at least partially define a gap, wherein the two electrodes detect a current upon movement of the polynucleotide comprising a plurality of nucleotides through the gap;

(b) subjecting the polynucleotide to movement through the gap;

(c) measuring the current with time across the gap using the two electrodes during movement of the polynucleotide through the gap, wherein the current is measured at a sampling rate greater than about 1 kHz;

(d) determining durations and current levels associated with the current measured in (c) to identify individual nucleotides of the plurality of nucleotides with time, wherein the individual nucleotides are identified by comparing the current levels to a reference value(s); and

(e) determining a sequence of the polynucleotide based on an identification of the individual nucleotides of the plurality of nucleotides,

wherein the gap is dimensioned such that the current is measured at the picoampere level under an applied voltage that is (i) greater than or equal to 0.25 V or (ii) less than or equal to 0.75 V.

2. The method of claim 1, wherein (b) comprises subjecting the polynucleotide to movement through the gap while applying a voltage between the two electrodes.

3. The method of claim 1, wherein the gap has a distance that is less than two times a molecular diameter of a given nucleotide of the plurality of nucleotides.

4. The method of claim 1, wherein the gap has a distance that is at least 0.5 times the molecular diameter of the given nucleotide of the plurality of nucleotides.

5. The method of claim 1, wherein (a)-(e) are performed in the absence of amplification and chemical modification of a precursor of the polynucleotide.

6. The method of claim 1, wherein the gap has a distance that is adjustable.

7. The method of claim 1, further comprising adjusting a distance of the gap at least partially mechanically.

8. The method of claim 1, further comprising amplifying the current measured in (c).

9. The method of claim 1, wherein the current is tunneling current.

11. The method of claim 1, wherein the applied voltage is between 0.25 V and 0.75V.

12. The method of claim 1, wherein the gap is in a break junction of the solid substrate.

10. A method for sequencing a polynucleotide, comprising:

(a) causing the polynucleotide to pass between electrodes, wherein the electrodes detect a current upon movement of the polynucleotide comprising a plurality of nucleotides between the electrodes;

(b) using the electrodes to continuously measure current with time within a measurement time period;

(c) identifying isolated current levels while continuously measuring the current with time, wherein at least a subset of the current levels are different upon comparison to a reference value(s); and

(d) associating each of the isolated current levels with individual nucleotides of the plurality of nucleotides, thereby identifying a sequence of the polynucleotide,

wherein a given nucleotide of the plurality of nucleotides is trapped between the electrodes for a specific duration while the current is continuously measured.

13. The method of claim 10, wherein the current is tunneling current.

14. The method of claim 10, wherein the electrodes are spaced apart by a distance that is within about 1 nanometer of a molecular diameter of a given nucleotide of the plurality of nucleotides.

15. The method of claim 14, wherein the distance is adjustable, and wherein the method further comprises adjusting the distance at least partially mechanically.

16. A method for sequencing a polynucleotide, comprising:

(a) causing the polynucleotide comprising a plurality of nucleotides to pass between electrodes separated by a distance comparable to a size of a given nucleotide of the plurality of nucleotides, wherein the electrodes detect a current upon movement of the polynucleotide between the electrodes;

(b) measuring the current upon movement of the polynucleotide between the electrodes to yield a current distribution with time;

(c) dividing the current distribution into a plurality of segments, wherein individual segments of the plurality of segments correspond to individual nucleotides of the plurality of nucleotides; and

(d) comparing the plurality segments to one or more reference values to identify the individual nucleotides of the plurality of nucleotides, thereby generating a sequence of the polynucleotide;

wherein in (b), the current is measured under an applied voltage that is (i) greater than or equal to 0.25 V or (ii) less than or equal to 0.75 V.

17. The method of claim 16, further comprising normalizing the current measured in (b).

18. The method of claim 17, wherein the current is normalized by dividing the current by a current value(s) of one or more standard nucleotides.

19. The method of claim 16, further comprising causing at least one reference sample comprising a plurality of reference nucleotides to pass between the electrodes and determining the one or more reference values, and wherein the polynucleotide and/or the at least one reference sample is passed between the electrodes multiple times.

20. The method of claim 16, wherein the current is tunneling current.

21. The method of claim 16, wherein the applied voltage is less than or equal to 0.75 V.

22. The method of claim 16, wherein the electrodes at least partially define a gap that is dimensioned such that the current is measured at the picoampere level under the applied voltage.

23. The method of claim 16, wherein the applied voltage is between 0.25 V and 0.75V.

24. A system for sequencing a polynucleotide, comprising:

a solid substrate comprising two electrodes that at least partially define a gap, wherein the two electrodes detect a current upon movement of the polynucleotide comprising a plurality of nucleotides through the gap, and wherein the gap is in a break junction of the solid substrate; and

a computing device that (i) measures the current with time across the gap using the two electrodes during movement of the polynucleotide through the gap, wherein the current is measured at a sampling rate greater than about 1 kHz, (ii) determines durations and current levels associated with the current to identify individual nucleotides of the plurality of nucleotides with time, and (iii) determines a sequence of the polynucleotide based on an identification of the individual nucleotides of the plurality of nucleotides,

wherein the gap is dimensioned such that the current is measured at the picoampere level under an applied voltage that is (i) greater than or equal to 0.25 V or (ii) less than or equal to 0.75 V.

25. The system of claim 24, wherein the gap has a distance that is within about 1 nanometer of a molecular diameter of a given nucleotide of the plurality of nucleotides.

26. The system of claim 24, wherein the gap has a distance that is less than two times a molecular diameter of a given nucleotide of the plurality of nucleotides.

27. The system of claim 26, wherein the gap has a distance that is at least 0.5 times the molecular diameter of the given nucleotide of the plurality of nucleotides.

28. The system of claim 24, wherein the applied voltage is between 0.25 V and 0.75V.

29. The system of claim 24, wherein the current is tunneling current.

30. The system of claim 24, wherein the gap has a distance that is adjustable.