Nanopore separation devices and methods of using same

• US 20060073489A1
• Filed: 10/05/2004
• Published: 04/06/2006
• Est. Priority Date: 10/05/2004
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Li, Gangqiang, Pittaro, Richard J.
• Application Number:
  US10/959,442
• Publication Number:
  US 20060073489A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• 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 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• 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 invention relates to nanopore-based separation devices and systems, and methods and kits for using these. The devices may be used to separate and evaluate biopolymers such as nucleic acids and proteins.

Claims

1. A method for separating biopolymers, comprising:

providing a plurality of biopolymers in a first reservoir connected to a second reservoir by a nanopore;

establishing an electrical field across the nanopore sufficient to translocate a biopolymer across the nanopore, thereby separating the biopolymer from other biopolymers; and

collecting the translocated biopolymer in the second reservoir or biopolymers in the first reservoir.

2. The method of claim 1, wherein the biopolymer comprises a nucleic acid molecule.

3. The method of claim 2, wherein the nucleic acid molecule is single-stranded.

4. The method of claim 2, wherein the nucleic acid is at least partially double-stranded.

5. The method of claim 1, wherein the biopolymer is bound to another molecule and the method comprises translocating bound biopolymer.

6. The method of claim 1, wherein the biopolymer is bound to another molecule and the method comprises translocating unbound biopolymer.

7. The method of claim 1, wherein the biopolymer comprises a polypeptide.

8. The method of claim 1, wherein an electric field is applied for a sufficient amount of time to translocate a biopolymer of a selected size.

9. The method of claim 1, wherein the plurality of biopolymers comprises at least two of:

polypeptides, DNA, RNA, and carbohydrates and the method comprises translocating biopolymers selected from the group consisting of polypeptides, DNA, RNA, and carbohydrates into the second reservoir.

10. The method of claim 8, further comprising collecting a plurality of biopolymers of the selected size in the second reservoir.

11. The method of claim 10, wherein the first and second reservoir are connected by a plurality of nanopores.

12. The method of claim 1, wherein the first reservoir comprises a plurality of subreservoirs, each subreservoir connected to the second reservoir by a nanopore.

13. The method of claim 12, wherein an electric field is applied independently to at least two of the nanopores.

14. The method of claim 12, wherein biopolymers of different characteristics are translocated through at least two of the nanopores.

15. The method of claim 14, wherein the different characteristics comprise different sizes.

16. The method of claim 1, further comprising removing the collected biopolymer from the first or second reservoir after the translocation.

17. The method of claim 1, comprising removing biopolymers in the first reservoir to a first container and removing biopolymers from the second reservoir to a second container after the translocation.

18. The method of claim 16, further comprising the step of determining the sequence of the removed biopolymers.

19. The method of claim 8, wherein the biopolymer is a nucleic acid and the biopolymer of the selected size is less than about 100 base pairs different in size from biopolymers remaining in the first reservoir.

20. The method of claim 8, wherein the biopolymer of the selected size is less than about 20 base pairs different in size from biopolymers remaining in the first reservoir.

21. The method of claim 4, wherein the biopolymer of the selected size is less than about 10 base pairs different in size from biopolymers remaining in the first reservoir.

22. The method of claim 1, further comprising monitoring current across the nanopore over time.

23. The method of claim 1, wherein the electric field is established by applying a voltage bias across the nanopore and the method further comprises reversing polarity of the applied voltage after a period of time sufficient to translocate a biopolymer of a selected size into the second reservoir.

24. The method of claim 23, wherein the voltage bias is controlled by a processor which receives input relating to passage of time after a current across the nanopore begins to decrease below a threshold amount, and provides instructions to a voltage source applying the voltage after a predetermined time sufficient to translocate the biopolymer of the selected size, to stop application of the voltage or to reverse polarity of the applied voltage.

25. The method of claim 24, wherein the processor accesses a memory and data relating to the predetermined time is stored in the memory.

26. The method of claim 24, wherein the processor is in communication with a user device and a user inputs data relating to the predetermined time into a display of the user device.

27. The method of claim 24, wherein the step of applying the voltage for the predetermined time is repeated one or more times.

28. The method of claim 1, wherein a plurality of biopolymers are translocated into the second reservoir through one or more nanopores connecting the first and second reservoir and wherein the method further comprises the step of concentrating translocated biopolymers.

29. The method of claim 1, wherein the method is used to separate allelic variants.

30. The method of claim 1, wherein the method is used to separate amplification products.

31. The method of claim 1, wherein the method is used to separate nucleic acid fragments generated by contacting nucleic acid samples with a nuclease.

32. The method of claim 1, wherein the nuclease is a restriction enzyme.

33. The method of claim 1, wherein the method is used to separate ligated nucleic acid molecules from non-ligated nucleic acid molecules.

34. The method of claim 1, wherein the method is used to separate and/or isolate cloning intermediates.

35. The method of claim 1, wherein the method is used to separate processed biopolymers from unprocessed biopolymers.

36. The method of claim 1, wherein the method is used to separate modified biopolymers from unmodified biopolymers.

37. The method of claim 1 wherein the method is used to separate spliced RNA molecules or cDNA copies thereof from unspliced or alternatively spliced molecules.

38. The method of claim 1, wherein the method is used to separate chromosomes of different size.

39. The method of claim 1, wherein the method is used to separate plasmid DNA from genomic DNA.

40. The method of claim 1, wherein the method is used to separate rearranged genes from non-rearranged genes.

41. The method of claim 5, wherein the method comprises collecting bound molecules, separating the molecules which are bound and determining the sequence of one or both of the bound molecules.

42. The method of claim 16, further comprising subjecting collected biopolymer to mass spectrometry.

43. The method of claim 1, wherein the second reservoir is connected to a third reservoir by a nanopore and the method comprises establishing an electrical field across the nanopore connecting the second and third reservoir sufficient to translocate a biopolymer from the second reservoir across the nanopore into the third reservoir.

44. A device comprising

a first reservoir connectable to a second reservoir by a nanopore;

a voltage source for generating an electric field across the nanopore; and

a

a processor in communication with the voltage source;

wherein the processor provides instructions to the voltage source to regulate the time period during which voltage is applied.

45. The device of claim 44, wherein the first reservoir is connectable to the second reservoir by a channel comprising the nanopore.

46. The device of claim 44, wherein the processor provides instructions to the voltage source to reverse polarity of an applied voltage after a period of time sufficient to translocate a biopolymer of a selected size across the nanopore.

47. The device of claim 44, wherein the device comprises a detector for measuring current over time and wherein the processor provides instructions to the voltage source to stop applying voltage or to reverse polarity of an applied voltage after a predetermined period of time after current decreases below a threshold level.

48. The device of claim 47, wherein the predetermined time is a time sufficient to translocate a biopolymer of a selected size.

49. The device of claim 44, wherein the first reservoir is connectable to the second reservoir by a plurality of nanopores.

50. The device of claim 44, wherein either or both first and second reservoirs comprise an outlet port for removing biopolymer.

51. The device of claim 44, wherein the first and/or second reservoir comprise channels.

52. The device of claim 44, wherein the device comprises a connecting channel in fluid communication with the second reservoir.

53. The device of claim 44, wherein the first reservoir comprises a plurality of subreservoirs, each subreservoir connectable to the second reservoir by a nanopore.

54. The device of claim 53, wherein a voltage may be independently applied across nanopores of at least two of the subreservoirs.

56. The device of claim 44, wherein the second reservoir comprises a plurality of reservoirs, each subreservoir connectable to the first reservoir by a nanopore.

57. The device of claim 56, wherein each of the subreservoirs comprises an outlet port for removing a sample.

58. The device of claim 52 wherein voltage across each nanopore is independently controlled.

59. The device of claim 44, wherein the device further comprises an electrode on either side of the nanopore.

60. A computer program product comprising a computer readable medium carrying program code for executing functions by the processor of claim 44.

61. A kit comprising a device of claim 44 and a biopolymer.

62. The kit of claim 58, wherein the biopolymer comprises a known size.

63. The kit of claim 58, wherein the kit comprises a plurality of biopolymers of known sizes.

64. A system comprising a device of claim 44, and a memory comprising a relational database that comprises data relating current impedance to size of biopolymers, and wherein the processor has access to the memory.

55. The device of 53, wherein device further comprises a third reservoir connectable to the second reservoir by a nanopore and a voltage may be applied independently across nanopores connecting first and second reservoirs and second and third reservoirs.