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

• US 9,194,838 B2
• Filed: 03/01/2011
• Issued: 11/24/2015
• Est. Priority Date: 03/03/2010
• Status: Active Grant

• Current Assignee: Osaka University
• Original Assignee: Osaka University
• Inventors: Taniguchi, Masateru, Tsutsui, Makusu, Yokota, Kazumichi, Kawai, Tomoji
• Primary Examiner(s): Ball, J. Christopher
• Application Number:
  US13/992,328
• Publication Number:
  US 20140300339A1
• Time in Patent Office: 1,729 Days
• US Class Current: 1/1
• 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 nucleic acid molecule, comprising:

(a) providing a solid substrate comprising a pair of electrodes that at least partially define a gap, wherein said gap has a distance that is less than a molecular diameter of said nucleic acid molecule;

(b) moving said nucleic acid molecule through said gap;

(c) detecting pulses of a tunnel current across said gap using said pair of electrodes, which pulses correspond to individual nucleic acid subunits of said nucleic acid molecule;

(d) calculating a modal value of current of said pulses detected in (c); and

(e) comparing said modal value calculated in (d) against a reference value to identify an individual nucleic acid subunit of said nucleic acid molecule.

2. The method of claim 1, further comprising normalizing said modal value.

3. The method of claim 2, wherein said modal value is normalized by dividing said modal value by a modal value of a standard nucleotide.

4. The method of claim 3, wherein the modal value of said standard nucleotide is generated by (i) passing a standard nucleotide between said pair of electrodes a plurality of times, (ii) detecting pulses of a tunnel current arising between the electrodes as the standard nucleotide passes through, and (iii) calculating a modal value of the current value of each pulse.

5. The method of claim 1, wherein said reference value is a normalized reference value.

6. The method of claim 1, further comprising repeating (c)-(e) with other pulses to identify other nucleic acid subunits of said nucleic acid molecule.

7. The method of claim 1, wherein the modal value is calculated by (i) generating a histogram expressing a relationship between current values of said individual pulse and a number of pulses corresponding to said current values, (ii) fitting said histogram to a given function, and (iii) deriving a peak value of said given function.

8. The method of claim 7, wherein said given function is a Gaussian function.

9. The method of claim 1, wherein said nucleic acid molecule is moved through said gap while applying a voltage between said electrodes.

10. The method of claim 1, wherein individual electrodes of said pair of electrodes comprise a noble metal.

11. The method of claim 1, wherein said gap has a distance that is from about 0.5 times to less than a molecular diameter of a given nucleic acid subunit of said nucleic acid molecule.

12. A method for nucleic acid sequencing, comprising:

(a) providing a solid substrate comprising a pair of electrodes that at least partially define a gap, wherein said gap has a distance that is less than a molecular diameter of said nucleic acid molecule, and wherein said pair of electrodes comprises individual electrodes that are adapted to detect individual nucleic acid subunits of said nucleic acid molecule upon the movement of said nucleic acid molecule through said gap;

(b) moving said nucleic acid molecule through said gap;

(c) while moving said nucleic acid molecule through said gap, detecting pulses of a tunnel current across said gap using said pair of electrodes, which pulses correspond to an individual nucleic acid subunit of said nucleic acid molecule; and

(d) using said pulses detected in (c), determining a nucleic acid sequence of said nucleic acid molecule.

13. The method of claim 12, wherein said individual electrodes comprise a noble metal.

14. The method of claim 12, further comprising, based on the pulses detected in (c), determining that (i) a nucleic acid subunit of said nucleic acid molecule is trapped between said electrodes, or (ii) said nucleic acid subunit has moved away from between said electrodes.

15. The method of claim 12, wherein (d) further comprises determining a dependence of said pulses on voltage applied across said gap, which dependence is indicative of an electron transport mechanism of each of said individual nucleic acid subunits.

16. The method of claim 12, wherein (d) further comprises (i) generating a histogram expressing a relationship between a current value of an individual pulse among said pulses and a number of pulses corresponding to said current value, (ii) fitting said histogram to a given function, and (iii) deriving a peak value of said given function.

17. The method of claim 16, further comprising identifying an individual nucleic acid subunit of said nucleic acid molecule based on said peak.

18. The method of claim 12, wherein said gap has a distance that is from about 0.5 times to less than a molecular diameter of a given nucleic acid subunit of said nucleic acid molecule.

19. A method for sequencing a nucleic acid molecule, comprising:

(a) providing a solid substrate comprising a pair of electrodes that at least partially define a gap, wherein said gap has a distance that is from about 0.5 times to less than a molecular diameter of a given nucleic acid subunit of said nucleic acid molecule;

(b) moving said nucleic acid molecule through said gap;

(c) while moving said nucleic acid molecule through said gap, detecting pulses of a current across said gap using said pair of electrodes;

(d) using a duration and current level associated with said pulses detected in (c), identifying individual nucleic acid subunits of said nucleic acid molecule; and

(e) from said nucleic acid subunits identified in (d), determining a nucleic acid sequence of said nucleic acid molecule.

20. The method of claim 19, wherein said individual nucleic acid subunits are identified by comparing said current level to a reference value.

21. The method of claim 19, wherein said nucleic acid molecule is moved through said gap while applying a voltage between said electrodes.

22. The method of claim 19, wherein individual electrodes of said pair of electrodes comprise a noble metal.

23. A method for sequencing a nucleic acid molecule, comprising:

(a) providing a solid substrate comprising a pair of electrodes that at least partially define a gap, wherein said gap has a distance that is less than a molecular diameter of said nucleic acid molecule;

(b) moving said nucleic acid molecule through said gap;

(c) while moving said nucleic acid molecule through said gap, detecting pulses of a tunnel current across said gap using said pair of electrodes;

(d) fitting said pulses to a given function that has a peak value of current, wherein said given function corresponds to an individual nucleic acid subunit of said nucleic acid molecule; and

(e) comparing said given function against a reference to identify said individual nucleic acid subunit of said nucleic acid molecule.

24. The method of claim 23, further comprising repeating (c)-(e) with other pulses to identify other nucleic acid subunits of said nucleic acid molecule.

25. The method of claim 23, wherein (d) comprises (i) generating a histogram expressing a relationship between current values of said pulses and a number of pulses corresponding to said current values, (ii) fitting said histogram to said given function, and (iii) deriving said peak value of said given function.

26. The method of claim 25, wherein said given function is a Gaussian function.

27. The method of claim 23, wherein individual electrodes of said pair of electrodes comprise a noble metal.

28. A method for sequencing a nucleic acid molecule, comprising (i) moving said nucleic acid molecule through a gap at least partially defined by an electrode pair in a solid substrate, which gap is dimensioned such that tunnel current across said gap is at the picoampere level under an applied voltage from about 0.25 V to 0.75 V, and (ii) reading signals indicative of said tunnel current at a frequency greater than about 1 kHz, thereby identifying individual nucleic acid subunits of said nucleic acid molecule.

29. The method of claim 28, wherein said signals include pulses of said tunnel current.

30. The method of claim 29, further comprising calculating a modal value of a current of an individual pulse among said pulses, and comparing said modal value to a reference value to identify an individual nucleic acid subunit of said nucleic acid molecule.

31. The method of claim 28, wherein said gap is dimensioned such that, with at least a portion of said nucleic acid molecule or individual nucleic acid subunits in said gap, a measured conductance is at the picoSiemens level.

32. The method of claim 28, wherein said gap has a distance that is less than a molecular diameter of said nucleic acid molecule.

33. The method of claim 28, wherein said gap has a distance that is from about 0.5 to 2 times a molecular diameter of a given nucleic acid subunit of said nucleic acid molecule.

34. A system for sequencing a nucleic acid molecule, comprising:

a solid substrate comprising a pair of electrodes that at least partially define a gap, wherein said gap has a distance that is less than a molecular diameter of said nucleic acid molecule; and

a calculation unit coupled to said pair of electrodes, wherein said calculation unit (i) uses said pair of electrodes to detect pulses of a tunnel current that correspond to individual nucleic acid subunits of said nucleic acid molecule, (ii) calculates a modal value of current of said pulses, and (iii) compares said modal value against a reference value to identify an individual nucleic acid subunit of said nucleic acid molecule.

35. The system of claim 34, wherein said pair of electrodes are spaced apart by a distance that is from about 0.5 times to less than a molecular diameter of a given nucleic acid subunit of said nucleic acid molecule.

36. The system of claim 34, wherein individual electrodes of said pair of electrodes comprise a noble metal.

37. The system of claim 34, wherein said calculation unit normalizes said modal value.

38. The system of claim 37, wherein said calculation unit normalizes said modal value by dividing said modal value by a modal value of a standard nucleotide.

39. The system of claim 34, wherein said reference value is a normalized reference value.

40. The system of claim 34, wherein said calculation unit repeats (i)-(iii) with other pulses to identify other nucleic acid subunits of said nucleic acid molecule.

41. The system of claim 34, wherein said calculation unit calculates said modal value by:

generating a histogram expressing a relationship between said current value of said individual pulse and a number of pulses corresponding to said current value;

fitting said histogram to a given function; and

deriving a peak value of said given function.

42. The system of claim 41, wherein said given function is a Gaussian function.