Microfluidic sequencing systems

• US 7,238,323 B2
• Filed: 12/05/2002
• Issued: 07/03/2007
• Est. Priority Date: 04/04/1997
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Inventors: Parce, John Wallace, Kopf-Sill, Anne R., Bousse, Luc J., Knapp, Michael
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/314,092
• Publication Number:
  US 20030104466A1
• Time in Patent Office: 1,671 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 422/68.1
• US Class Current: 422/68.1
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/0262 using touch-off at substrate or container
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502784 : specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• 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 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: G01N 2035/1053 : for separating part of the liquid, e.g. filters, extraction phase
• CPC Class Codes: G01N 2035/1058 : for mixing

Abstract

Integrated systems, apparatus, software, and methods for performing biochemical analysis, including DNA sequencing, genomic screening, purification of nucleic acids and other biological components and drug screening are provided. Microfluidic devices, systems and methods for using these devices and systems for performing a wide variety of fluid operations are provided. The devices and systems of are used in performing fluid operations which require a large number of iterative, successive or parallel fluid manipulations, in a microscale, or sealed and readily automated format.

Claims

1. A sequencing apparatus comprising:

a body having a top portion, a bottom portion and an interior portion;

the interior portion comprising at least two intersecting channels, wherein at least one of the two intersecting channels has at least one cross sectional dimension between about 0.1 μ

m and 500 μ

m;

an electrokinetic fluid direction system for moving a first sequencing reagent through at least one of the two intersecting channels;

a source of sequencing primers;

an electropipettor for introducing sequencing primers from the source of sequencing primers to the at least two intersecting channels;

a mixing zone fluidly connected to the at least two intersecting channels for mixing the first sequencing reagent with a second sequencing reagent;

a size separation zone fluidly connected to the mixing zone for separating sequencing products by size, thereby providing the sequence of a target nucleic acid; and

a computer having computer-readable code for selecting a sequencing primer.

2. The apparatus of claim 1, farther comprising a sequence detector for reading the sequence of the target nucleic acid.

3. The apparatus of claim 1, wherein the source of sequencing primers comprises a set of about 4,096 primers.

4. The apparatus of claim 3, wherein the primers are a set of approximately all possible 6 mers.

5. A nucleic acid sequencing system, comprising:

a body structure comprising a plurality of microfluidic channels, wherein the body structure is fluidly coupled to a pipettor capillary that extends outward from the body structure;

a source of one or more template nucleic acid, which template nucleic acid source is capable of being fluidly coupled to at least a first one of the microfluidic channels;

a source of one or more sequencing reagent, which sequencing reagent source is capable of being fluidly coupled to the at least first microfluidic channel;

a detector; and

,

a fluid flow controller that flows the template nucleic acid and the one or more sequencing reagent into contact in the at least first microfluidic channel, whereby one or more sequencing product is formed, wherein the detector detects the one or more sequencing product, thereby determining at least a subsequence of the template nucleic acid.

8. The nucleic acid sequencing system of claim 5, 6, or 7 wherein the plurality of microfluidic channels are disposed within the body structure.

10. The nucleic acid sequencing system of claim 5 wherein the pipettor capillary is an electropipettor.

11. The nucleic acid sequencing system of claim 5, 6 or 7 wherein the source of the template nucleic acid or the source of the sequencing reagent, or both, is one or more well in or on the body structure.

12. The nucleic acid sequencing system of claim 5, 6 or 7 wherein the source of the template nucleic acid or the source of the sequencing reagent, or both, is selected from the group consisting of:

a test tube, a plate, a microtiter plate, a 96 well plate, 384 well plate, an array, and an array of dried materials.

13. The nucleic acid sequencing system of claim 5, wherein the capillary accesses the source of the template nucleic acid, or the source of the sequencing reagent, or both.

14. The nucleic acid sequencing system of claim 13, wherein the system comprises system instructions that control fluid expulsion from the capillary to the source of the template nucleic acid, or the source of the sequencing reagent, or both, thereby hydrating the template nucleic acid, or the sequencing reagent, or both.

15. The nucleic acid sequencing system of claim 14, wherein the system instructions further direct the capillary to draw the hydrated template nucleic acid, or the sequencing reagent, or both, into the body structure trough the capillary.

18. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing reagent is a primer, wherein the primer is delivered to the at least first microfluidic channel from a set of primers comprising at least about 70% of all possible primers of a given length.

19. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing reagent is a primer, wherein the primer is delivered to the at least first microfluidic channel from a set of primers comprising at least about 90% of all possible primers of a given length.

20. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing reagent is a primer, wherein the primer is delivered to the at least first microfluidic channel from a set of primers comprising all possible primers of a given length.

21. The nucleic acid sequencing system of claim 5 6 or 7, wherein the source of the sequencing reagent comprises a plurality of primers in an array.

22. The nucleic acid sequencing system of claim 21, wherein the primers are at specified spatial locations on the array.

23. The nucleic acid sequencing system of claim 22, wherein each of the primers is assigned a physical array address, wherein the system comprises system instructions for accessing a primer at a specified physical array address.

24. The nucleic acid sequencing system of claim 21, wherein the system comprises at least about 3,000 primers.

25. The nucleic acid sequencing system of claim 21, wherein the system comprises at least about 4,096 primers.

26. The nucleic acid sequencing system of claim 5 or 7, wherein the system comprises at least about 70% of all possible primers that are a given number of nucleotides in length.

27. The nucleic acid sequencing system of claim 21, wherein the system comprises at least about 70% of all possible primers that are a given number of nucleotides in length and wherein the given length is between about 4 and about 10 nucleotides.

28. The nucleic acid sequencing system of claim 21, wherein the system comprises at least about 90% of all possible primers that are a given number of nucleotides in length.

29. The nucleic acid sequencing system of claim 28, wherein the given length is between about 4 and about 10 nucleotides.

30. The nucleic acid sequencing system of claim 21, wherein the primers comprise modular primers tat are complementary to adjacent sites on the template nucleic acid.

31. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the template nucleic acid comprises one or more of:

an RNA, a DNA, a cloned nucleic acid, a plasmid, a lambda phage, a cosmid, a yeast artificial chromosome (YAC), a P1 artificial chromosome, a MAC, a PAC, or a Bacterial Artificial Chromosome (BAC).

32. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the template nucleic acid is attached to a bead.

33. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the template nucleic acid is end labeled.

34. The nucleic add sequencing system of claim 33, wherein the one or more sequencing product comprises a cleavage product of the end-labeled template.

35. The nucleic acid sequencing system of claim 33, wherein the one or more sequencing product comprises a primer hybridized to the end-labeled template nucleic acid.

36. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the template nucleic acid is unlabeled.

37. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing product comprises a template-dependent primer extension product of a primer hybridized to the template nucleic acid.

38. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing product comprises a labeled primer hybridized to the template nucleic acid.

39. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing reagent comprises one or more of:

a primer that is complementary to a subsequence of the template nucleic acid, a perfectly matched sequencing by hybridization primer, an imperfectly matched sequencing by hybridization primer, a modular primer, a labeled primer, an unlabeled primer, a dNTP, a labeled dNTP, a ddNTP, an enzyme, a polymerase, a thermostable polymerase, an E. coli DNA polymerase, an E. coli DNA pol I, a Klenow fragment, a T7 DNA polymerase, a Taq polymerase, a Pfu DNA polymerase, a Bst polymerase, a Vent polymerase, magnesium, potassium, sodium, NaOH, or a chemical that degrades the template nucleic acid.

40. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing reagent is attached to a bead.

41. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing reagent comprises a primer that is complementary to the template nucleic acid, dNTPs, ddNTPs and a polymerase.

42. The nucleic acid sequencing system of claim 41, wherein the one or more sequencing product is a primer extension product that comprises a ddNTP at one terminus of the product.

43. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing reagent comprises an exonuclease and forming the sequencing products comprise an exonuclease digested nucleic acid.

44. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing product comprises a boronated nuclease resistant nucleotide.

45. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing product comprises one or more radioactive moiety.

46. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing product comprises one or more fluorescent moiety.

47. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing product comprises a plurality of fluorescent moieties.

48. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the one or more sequencing product comprises four different fluorescent moieties.

49. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the detector comprises a fluorescence detector that is capable of detecting at least four different color emissions.

50. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the detector comprises a spectrophotometer, a fluorescent detector, a microscope, a fluorescence polarization detector, or a scintillation counting device.

51. The nucleic acid sequencing system of claim 5, 6 or 7, wherein she detector is coupled to a computer, which computer comprises instructions that convert a signal from the detector into the template nucleic acid subsequence.

52. The nucleic acid sequencing system of claim 51, wherein the computer comprises instructions that compile the template nucleic acid subsequence with one or more additional subsequence of the template nucleic acid to provide a sequence of the template nucleic acid.

53. The nucleic acid sequencing system of claim 51, wherein the computer comprises instructions that determine whether there is additional sequence to be obtained from the template nucleic acid.

54. The nucleic acid sequencing system of claim 53, wherein the computer provides a user readable output to indicate that sequencing of the template is complete.

55. The nucleic acid sequencing system of claim 51, wherein the computer is operably linked to the fluid flow controller, wherein the computer comprises instructions that, based upon the nucleic acid subsequence, directs the fluid flow controller to flow one or more additional sequencing reagent into contact with the template nucleic acid.

56. The nucleic acid sequencing system of claim 51, wherein the computer is operably linked to the fluid flow controller, wherein the computer comprises instructions that, based upon the nucleic acid subsequence, directs the fluid flow controller to flow one or more additional template nucleic acid into the body structure.

57. The nucleic acid sequencing system of claim 56, wherein the computer selects one or more sequencing primer for one or more sequencing reaction, wherein the sequencing primer is complementary to the subsequence of the template nucleic acid, wherein the computer instructs the fluid flow controller to access the sequencing primer.

58. The nucleic acid sequencing system of claim 57, wherein the sequencing primer is a primer in an array of primers and the fluid flow controller accesses the sequencing primer from the array.

59. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the fluid flow controller comprises a voltage controller that applies a voltage to one or more of the microfluidic channels.

60. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the fluid flow controller comprises a pump that applies a pressure to one or more of the microfluidic channels.

61. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the system comprises a fluidic interface that transports materials into or out of the body structure.

63. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the fluid flow controller directs flow of the template nucleic acid from the template nucleic acid source through a second microfluidic channel to a mixing region within the first microfluidic channel and flow of the sequencing reagent from the reagent source through a third microfluidic channel into the mixing region.

64. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the system comprises a robotic armature that moves the body structure into proximity to the template nucleic acid source, or to the sequencing reagent source.

65. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the system comprises a robotic armature that moves the template nucleic acid source, or the sequencing reagent source into proximity to the body structure.

66. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the system comprises instructions that provide for sequencing of the template nucleic acid by primer walking.

67. The nucleic acid sequencing system of claim 5, 6 or 7, wherein the system comprises instructions that direct the fluid flow controller to:

flow the template nucleic acid from the template nucleic acid source through a at least a second microfluidic channel to a mixing region within the first microfluidic channel;

flow a first nucleic acid sequencing reagent from a first reagent source into the mixing region;

flow a second nucleic acid sequencing reagent from a second reagent source into the mixing region.

68. The nucleic acid sequencing system of claim 67, wherein the system comprises instructions that select at least a third sequencing reagent based upon the subsequence of the template nucleic acid, or that direct selecting a second template nucleic acid for sequencing based upon the subsequence of the template nucleic acid.

69. The nucleic acid sequencing system of claim 68, wherein the template nucleic acid and the second template nucleic acid are both genomic clones of an organism.

70. The nucleic acid sequencing system of claim 68, wherein the third sequencing reagent is a primer that is complementary to a portion of the subsequence of the template nucleic acid.

74. The nucleic acid sequencing system of claim 5, 6 or 7, further comprising a fluid heater that heats fluid in at least one microfluidic channel in the body structure.

75. The nucleic acid sequencing system of claim 74, wherein the fluid heater comprises a voltage controller or a current controller.

76. The nucleic acid sequencing system of claim 74, wherein the fluid heater passes electrical current through a fluid in the at least one microfluidic channel, thereby heating the fluid.

77. The nucleic acid sequencing system of claim 74, wherein the system comprises instructions that cause the fluid heater to thermocycle the fluid in the at least one microfluidic channel.

78. The nucleic acid sequencing system of claim 77, wherein the system instructions repetitively thermocycle the fluid in the at least one microfluidic channel.

6. A nucleic acid sequencing system, comprising:

a body structure comprising a plurality of microfluidic channels;

a source of one or more template nucleic acid, which template nucleic acid source is capable of being fluidly coupled to at least a first one of the microfluidic channels;

a source of one or more sequencing reagent, which sequencing reagent source is capable of being fluidly coupled to the at least first microfluidic channel;

wherein the source of one or more sequencing reagent comprises a set of sequencing primers, the set of primers comprising at least about 70% of all possible primers of a given length;

a detector; and

,

a fluid flow controller that flows the template nucleic acid and the one or more sequencing reagent into contact in the at least first microfluidic channel, whereby one or more sequencing product is formed, wherein the detector detects the one or more sequencing product, thereby determining at least a subsequence of the template nucleic acid.

9. The nucleic acid sequencing system of claim 6 or 7, wherein the body structure is fluidly coupled to a pipettor capillary that extends outward from the body structure.

16. The nucleic acid sequencing system of claim 6, wherein the set is located in a microfluidic device that comprises the plurality of microfluidic channels.

17. The nucleic acid sequencing system of claim 6, wherein the set is located on a substrate, wherein members of the set are accessed using a microfluidic device that comprises the plurality of microfluidic channels.

62. The nucleic acid sequencing system of claim 61, 6 or 7, wherein the fluid flow controller comprises or is operably coupled to the fluidic interface.

7. A nucleic acid sequencing system, comprising:

a body structure comprising a plurality of microfluidic channels;

a source of one or more template nucleic acid, which template nucleic acid source is capable of being fluidly coupled to at least a first one of the microfluidic channels;

a source of one or more sequencing reagent, which sequencing reagent source is capable of being fluidly coupled to the at least first microfluidic channel;

a detector; and

a fluid flow controller that flows the template nucleic acid and the one or more sequencing reagent into contact in the at least flint microfluidic channel, whereby one or more sequencing product is formed, wherein the detector detects the one or more sequencing product, thereby determining at least a subsequence of the template nucleic acid,

wherein the system comprises a sequencing product separation channel into which the one or more sequencing product is flowed and separated by size, or charge, or size and charge, into a plurality of separated sequencing products.

71. The nucleic acid sequencing system of claim 7, wherein the detector is positioned to detect the separated sequencing products.

72. The nucleic acid sequencing system of claim 7, wherein the separation channel comprises chromatographic material.

73. The nucleic acid sequencing system of claim 7, wherein the separation channel comprises a polymer.