Droplet-based nucleic acid amplification method and apparatus

• US 20090291433A1
• Filed: 12/15/2006
• Published: 11/26/2009
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Paik, Philip Y., Pollack, Michael G.
• Granted Patent: US 7,851,184 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention relates to a droplet-based nucleic acid amplification method and apparatus. According to one embodiment, a method of amplifying a nucleic acid in a biological sample is provided, wherein the method includes: (a) providing a system comprising a droplet microactuator electronically coupled to and controlled by a processor capable of executing instructions, the droplet microactuator comprising: (i) a sample potentially comprising a target nucleic acid; (ii) a substrate comprising electrodes for conducting droplet operations; and (iii) one or more temperature control means arranged in proximity with one or more of the electrodes for heating a region of the droplet microactuator such that a droplet can be transported into the region for heating; (b) using droplet operations to combine on the droplet microactuator one or more amplification reagent droplets and one or more sample droplets to yield an amplification-ready droplet; and (c) thermal cycling the amplification-ready droplet sufficient to result in amplification of a target nucleic acid when present in the amplification-ready droplet.

Claims

1. A method of amplifying a nucleic acid in a biological sample, the method comprising:

(a) providing a system comprising a droplet microactuator electronically coupled to and controlled by a processor capable of executing instructions, the droplet microactuator comprising;

(i) a sample potentially comprising a target nucleic acid;

(ii) a substrate comprising electrodes for conducting droplet operations; and

(iii) one or more temperature control means arranged in proximity with one or more of the electrodes for heating a region of the droplet microactuator such that a droplet can be transported into the region for heating;

(b) using droplet operations to combine on the droplet microactuator one or more amplification reagent droplets and one or more sample droplets to yield an amplification-ready droplet; and

(c) thermal cycling the amplification-ready droplet sufficient to result in amplification of a target nucleic acid when present in the amplification-ready droplet.

2. The method of claim 1 wherein step 1(b) comprises:

(a) transporting one or more amplification reagent droplets from a reservoir on the droplet microactuator;

(b) transporting one or more sample droplets from a reservoir on the droplet microactuator; and

(c) merging one or more amplification reagent droplets with one or more sample droplets to yield the amplification-ready droplet.

3. The method of claim 2 further comprising:

(a) loading one or more amplification reagent droplets into a reservoir on the droplet microactuator by flowing the one or more amplification reagent droplets into the reservoir from a locus exterior to the droplet microactuator; and

/or

(b) loading one or more sample droplets into a reservoir on the droplet microactuator by flowing the one or more amplification reagent droplets into the reservoir from a locus exterior to the droplet microactuator.

4. The method of claim 1 further comprising:

(a) loading one or more amplification reagent droplets onto the droplet microactuator by flowing the one or more amplification reagent droplets onto the droplet microactuator from a locus exterior to the droplet microactuator; and

/or

(b) loading one or more sample droplets onto the droplet microactuator by flowing the one or more sample droplets onto the droplet microactuator from a locus exterior to the droplet microactuator.

5. The method of claim 1 wherein the amplification-ready droplet has a volume which ranges from about 1 mL to about 10 μ

L.

6. The method of claim 1 wherein the amplification-ready droplet has a volume which ranges from about 1 mL to about 1 μ

L.

7. The method of claim 1 wherein the amplification-ready droplet has a volume which is less than about 100 mL.

8. The method of claim 1 further comprising detecting the presence or absence of amplified target nucleic acid in the amplification-ready droplet during or after thermal cycling.

9. The method of claim 1 further comprising dispensing a droplet from the amplification-ready droplet during thermal cycling.

10. The method of claim 1 further comprising quantifying the quantity of amplified target nucleic acid in the amplification-ready droplet during or after thermal cycling.

11. The method of claim 10 further comprising extrapolating the quantity of target nucleic acid in the amplification-ready droplet prior to thermal cycling.

12. The method of claim 1 wherein step 1(c) is accomplished in the amplification-ready droplet in proximity to a heater by changing the temperature of the heater.

13. The method of claim 1 wherein step 1(c) is accomplished by transporting the amplification-ready droplet into and out of proximity with one or more heaters.

14. The method of claim 1 further comprising conducting droplet operations selected to immobilize amplified nucleic acid on a surface having an affinity for the amplified nucleic acid.

15. The method of claim 14 wherein the surface comprises a surface of the droplet microactuator.

16. The method of claim 14 wherein the surface comprises one or more beads.

17. The method of claim 14 wherein the droplet operations comprise subjecting the amplified target nucleic acid to denaturing conditions to render it single-stranded prior to contact with the surface.

18. The method of claim 14 wherein the droplet operations comprise merging the amplification-ready droplet comprising amplified target nucleic acid with a droplet comprising the beads.

19. The method of claim 14 wherein the amplified target nucleic acid is biotinylated and the beads comprise streptavidin bound thereto.

20. The method of claim 14 further comprising conducting droplet operations sufficient to:

(a) denature the amplified nucleic acid to provide single-stranded nucleic acid; and

(b) contact the single-stranded nucleic acid with a substrate having an affinity for the single-stranded nucleic acid thereby immobilizing the single-stranded nucleic acid on the substrate.

21. The method of claim 14 further comprising executing a droplet-based washing protocol to separate unbound components of the amplification-ready droplet from the surface.

22. The method of claim 21 wherein the droplet-based washing protocol comprises conducting droplet operations to:

(a) bring a wash droplet into contact with the surface; and

(b) separate the wash droplet from the surface thereby carrying away unbound substances from the surface.

23. The method of claim 16 further comprising executing a droplet-based washing protocol to separate unbound components of the amplification-ready droplet from the beads.

24. The method of claim 23 wherein the droplet-based washing protocol comprises:

(a) immobilizing the beads in the droplet;

(b) conducting droplet operations to merge a wash droplet with the droplet comprising the beads to yield a merged droplet; and

(c) conducting droplet operations to separate a droplet from the merged droplet thereby carrying away unbound substances from the beads.

25. The method of claim 24 further comprising repeating steps (a) to (c) until a predetermined concentration of unbound substances is achieved.

26. The method of claim 24 further comprising a step (d) comprising releasing the immobilized beads.

27. The method of claim 26 further comprising repeating steps (a) to (d) until a predetermined concentration of unbound substances is achieved.

28. The method of claim 24 wherein the beads are magnetically responsive and the immobilizing step is achieved by using a magnet to immobilize the magnetically responsive beads in the droplet.

29. The method of claim 24 wherein the beads are not significantly responsive to a magnetic field and the immobilizing step is achieved by using a physical object to block transport of such beads during a droplet operation.

30. The method of claim 23 wherein the droplet-based washing protocol comprises:

(a) conducting droplet operations to merge a wash droplet with the droplet comprising the beads to yield a merged droplet;

(b) immobilizing the beads in the merged droplet; and

(c) conducting droplet operations to separate a droplet from the merged droplet thereby carrying away unbound substances from the beads.

31. The method of claim 30 further comprising repeating steps (a) to (c) until a predetermined concentration of unbound substances is achieved.

32. The method of claim 30 further comprising a step (d) comprising releasing the immobilized beads.

33. The method of claim 32 further comprising repeating steps (a) to (d) until a predetermined concentration of unbound substances is achieved.

34. The method of claim 30 wherein the beads are magnetically responsive and the immobilizing step is achieved by using a magnet to immobilize the magnetically responsive beads in the droplet.

35. The method of claim 30 wherein the beads are not magnetically responsive and the immobilizing step is achieved by using a physical object to block transport of the magnetically responsive beads during a droplet operation.

36. The method of claim 23 further comprising executing droplet operations selected to effect an elution protocol for freeing immobilized nucleic acid from the surface.

37. The method of claim 1 further comprising quantifying amplified target nucleic after a predetermined number of cycles.

38. The method of claim 1 further comprising stopping the thermal cycling when:

(a) a predetermined quantity of target nucleic acid is detected;

or

(b) a predetermined quantity of target nucleic acid is not detected after a predetermined number of cycles.

39. The method of claim 1 further comprising transporting a droplet comprising amplified nucleic acid for further processing.

40. The method of claim 1 further comprising detecting one or more target nucleic acids.

41. The method of claim 40 further comprising providing a user output indicating the presence or absence of one or more target nucleic acids

42. The method of claim 40 wherein the target nucleic acid comprises a diagnostic nucleic acid.

43. The method of claim 1 wherein step 0 is preceded by flowing one or more reagents and/or one or more samples from a locus exterior to the droplet microactuator onto the droplet microactuator.

44. The method of claim 43 wherein the external reservoir is located on a cartridge which is coupled to the droplet microactuator in a manner which establishes a flow path from the external reservoir onto the droplet microactuator.

45. The method of claim 43 wherein the external reservoir is located on a cartridge which is coupled to the droplet microactuator in a manner which establishes a flow path from the external reservoir into a reservoir on the droplet microactuator.

46. A method of amplifying a nucleic acid on a droplet microactuator, the method comprising repeatedly transporting an amplification-ready droplet having a volume which does not exceed 50 mL through one or more heated zones on a substrate.

47. The method of claim 46 wherein the transporting comprises electric field-mediated transporting.

48. The method of claim 46 wherein the transporting comprises electrode-mediated transporting.

49. The method of claim 46 wherein the transporting comprises electrowetting-mediated transporting.

50. The method of claim 46 wherein the transporting comprises dielectrophoresis-mediated transporting.

51. The method of claim 46 wherein the amplification-ready droplet is substantially lacking in surfactant.

52. A method of conducting a droplet operation at an elevated temperature, the method comprising:

(a) providing a droplet microactuator;

(b) heating a droplet thereon to a temperature exceeding about 25°

C. to yield a heated droplet; and

(c) conducting a droplet operation on the heated droplet.

53. The method of claim 52 wherein step 52(b) comprises heating the droplet to a temperature exceeding about 30°

C.

54. The method of claim 52 wherein step 52(b) comprises heating the droplet to a temperature exceeding about 50°

C.

55. The method of claim 52 wherein step 52(b) comprises heating the droplet to a temperature exceeding about 75°

C.

56. The method of claim 52 wherein the droplet operation is electric field-mediated.

57. The method of claim 52 wherein the droplet operation is electrode-mediated.

58. The method of claim 52 wherein the droplet operation is electrowetting-mediated.

59. The method of claim 52 wherein the droplet operation is dielectrophoresis-mediated.

60. The method of claim 52 wherein the droplet comprises a droplet in an incubation step of a droplet-based assay protocol.

61. The method of claim 56 wherein the droplet-based assay protocol comprises an affinity-based assay protocol.

62. A droplet microactuator made using a fluorescing material and comprising a detection region for detecting a fluorescence signal from a droplet, which detection region is coated with a light absorbing, low fluorescence or non-fluorescing material.

63. The droplet microactuator of claim 62 wherein the fluorescing material comprises a dielectric material.

64. The droplet microactuator of claim 62 wherein the coating comprises a metal coating.

65. The droplet microactuator of claim 62 wherein the coating comprises a copper coating.

66. The droplet microactuator of claim 62 wherein the coating comprises a black coating.

67. The droplet microactuator of claim 62 wherein the coating comprises a copper coating comprising a gold finish applied thereto.