Nanopore-based single molecule characterization

• US 9,121,059 B2
• Filed: 12/21/2011
• Issued: 09/01/2015
• Est. Priority Date: 12/22/2010
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Genia Technologies, Inc. (Roche Holding AG)
• Inventors: Davis, Randall W., Chen, Roger J. A.
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/333,915
• Publication Number:
  US 20120160688A1
• Time in Patent Office: 1,350 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/447 using electrophoresis
• 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
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis

Abstract

The present invention relates to a method of using nanopore to obtain sequence information of an unknown structure (unknown DNA) in a ss test DNA. The method comprises using speed bump to stall the ss test DNA in the nanopore at random positions of the ss test DNA to obtain sequence information of each and every nucleotides of the unknown DNA, and to construct the whole sequence of the unknown DNA. The present invention also relates to a novel method of trapping a ss test DNA in a nanopore using two bulky structures formed under different conditions (e.g. different temperature), and the bulky structures are able to keep the ss test DNA trapped in a nanopore at a working temperature.

Claims

1. A method for manipulating a nucleic acid molecule in a nanopore, the method comprising:

(a) forming a first bulky structure on a first end of a nucleic acid molecule at a first temperature;

(b) applying a first electrical potential to flow the nucleic acid molecule through a nanopore; and

(c) forming a second bulky structure on a second end of the nucleic acid molecule at a second temperature.

2. The method of claim 1, wherein, said nucleic acid molecule is a single-stranded nucleic acid molecule, and wherein said method further comprises prior to (a):

(a) providing a double-stranded nucleic acid molecule;

(b) ligating a first pre-bulky structure to a first end of the double-stranded nucleic acid molecule; and

(c) denaturing the double-stranded nucleic acid molecule of (f) to produce said single-stranded nucleic acid molecule.

3. The method of claim 2, wherein (e) further comprises ligating a second pre-bulky structure to a second end of the double-stranded nucleic acid molecule.

4. The method of claim 3, wherein the first pre-bulky structure and/or the second pre-bulky structure comprises a direction identifier.

5. The method of claim 3, wherein the second pre-bulky structure has a sequence of SEQ ID NO. 1.

6. The method of claim 1, further comprising, subsequent to (c), obtaining a first set of electrical signals while applying a second electrical potential to reverse the flow of the single-stranded nucleic acid molecule until the single-stranded nucleic acid molecule is stopped by the second bulky structure of step (c).

7. The method of claim 6, further comprising obtaining a second set of electrical signals while applying a third electrical potential to reverse the flow of the single-stranded nucleic acid molecule until the single-stranded nucleic acid molecule is stopped by the first bulky structure.

8. The method of claim 7, wherein said obtaining said first set of electrical signals and obtaining said second set of electrical signals are repeated.

9. The method of claim 8, wherein said obtaining said first set of electrical signals and obtaining said second set of electrical signals are repeated at least 10 times.

10. The method of claim 7, wherein the second and third electric potentials are the same.

11. The method of claim 7, wherein the second and third electric potentials are different.

12. The method of claim 7, wherein the second and/or third electric potentials are continuously changing.

13. The method of claim 6, wherein said first set of electrical signals are obtained at a third temperature.

14. The method of claim 13, wherein the first temperature is greater than the second temperature, and the second temperature is greater than the third temperature.

15. The method of claim 13, wherein the third temperature is from about −

10 to 20°

C.

16. The method of claim 1, wherein the first temperature is greater than the second temperature.

17. The method of claim 1, wherein said single-stranded nucleic acid molecule is contacted with one or more speed bumps.

18. The method of claim 17, wherein the one or more speed bumps comprise up to 12 nucleic acid bases.

19. The method of claim 17, wherein the one or more speed bumps comprise locked nucleotides, universal bases, dideoxy-nucleosides at the 3′

end and/or the 5′

end, morpholinos, glycol nucleotides, abasic nucleotides, methylated nucleobases, non-binding base mimics, or any combination thereof.

20. The method of claim 17, wherein an individual speed bump of the one or more speed bumps forms duplex sections with the single-stranded nucleic acid molecule, and the duplex sections have about the same melting temperatures.

21. The method of claim 17, wherein said one or more speed bumps are provided in a random speed bump pool.

22. The method of claim 21, wherein the random speed bump pool comprises speed bump trains formed by linking multiple speed bumps with non-binding linkers.

23. The method of claim 21, wherein said first bulky structure and/or said second bulky structure comprise non-binding structures that do not bind to the random speed bump pool.

24. The method of claim 23, wherein said non-binding structures are selected from the group consisting of isodG, isodC, abasic site and combinations thereof.

25. The method of claim 21, wherein said random speed bump pool comprises speed bumps having universal bases at a 3′

end and/or a 5′

end.

26. The method of claim 1, wherein the single-stranded nucleic acid molecule has a length from about 5 to 10,000 bases.

27. The method of claim 1, wherein the nanopore is an alpha hemolysin nanopore.

28. The method of claim 1, wherein said nanopore is in a membrane disposed adjacent or in proximity to an electrode.

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

(a) providing a chip comprising at least one nanopore in a lipid bilayer membrane that is disposed adjacent or in proximity to an electrode, wherein said electrode is adapted to detect said nucleic acid molecule or a portion thereof;

(b) threading the nucleic acid molecule into the nanopore at a first temperature;

(c) trapping the nucleic acid molecule in the nanopore at a second temperature; and

(d) detecting one or more nucleic acid bases of the nucleic acid molecule while the nucleic acid molecule is trapped in the nanopore.

30. The method of claim 29, wherein the first temperature is greater than the second temperature.

31. The method of claim 29, wherein (d) is performed at a third temperature, and wherein the third temperature is lower than the first temperature and the second temperature.

32. The method of claim 29, wherein said detecting in (d) further comprises applying an electrical potential to reverse the flow of the nucleic acid molecule and obtaining a set of electrical signals.

33. The method of claim 32, wherein the nucleic acid molecule further comprises a bulky structure at an end of the nucleic acid molecule, and the flow of the nucleic acid molecule through said nanopore is stopped by the bulky structure.

34. The method of claim 33, further comprising applying another electrical potential to reverse the flow of the nucleic acid molecule and obtaining another set of electrical signals.

35. The method of claim 34, wherein the nucleic acid molecule further comprises another bulky structure at another end of the nucleic acid molecule, and the flow of the nucleic acid molecule through the nanopore is stopped by said another bulky structure.

36. The method of claim 29, wherein said nucleic acid molecule is trapped with the aid of a first bulky structure at a first portion of the nucleic acid molecule and a second bulky structure at a second portion of the nucleic acid molecule.

37. The method of claim 36, wherein trapping the nucleic acid molecule in (d) further comprises:

i. forming said first bulky structure on said first portion of a nucleic acid molecule at the first temperature;

ii. applying an electrical potential to thread the nucleic acid molecule into the nanopore; and

iii. forming said second bulky structure on said second portion of the nucleic acid molecule at the second temperature.

38. The method of claim 37, wherein the first temperature is greater than the second temperature.