Closed-loop biochemical analyzers

• US 6,235,471 B1
• Filed: 04/03/1998
• Issued: 05/22/2001
• Est. Priority Date: 04/04/1997
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Parce, John Wallace, Kopf-Sill, Anne R., Bousse, Luc J., Knapp, Michael
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US09/054,962
• Time in Patent Office: 1,145 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/283.1, 536/24.33
• US Class Current: 435/6.19
• CPC Class Codes: B01F 33/30 Micromixers
• 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 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 2300/18 Means for temperature control
• CPC Class Codes: B01L 2300/1833 using electrical currents in the sample itself
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0262 using touch-off at substrate or container
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• 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
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027

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 method of sequencing a nucleic acid comprising:

providing a target nucleic acid, a first sequencing primer, a polymerase, ddNTPs, and ddNTPs;

mixing the target nucleic acid, the first sequencing primer, the polymerase, the dNTPs, acid the ddNTPs in a microfluidic device under conditions permitting target dependent polymerization of the dNTPs, thereby providing polymerization products; and

,

separating the polymerization products by size in the microfluidic device to provide a first sequence of the target nucleic acid.

2. The method of claim 1, wherein a second sequencing primer is selected based upon the sequence of the target nucleic acid and the second sequencing primer is mixed with the target nucleic acid in a microfluidic device under conditions permitting target dependent elongation of the selected second sequencing primer thereby providing polymerization products which are separated by size in the microfluidic device to provide a second sequence of the target nucleic acid.

3. The method of claim 1, wherein the first sequencing primer is selected from a large set of sequencing primers which comprises a primer having a sequence complementary to the target nucleic acid.

4. The method of claim 3, wherein the large set comprises at least about 70% of all possible sequencing primers for a given length, wherein the length is between about 4 and about 10.

5. The method of claim 3, wherein the large set comprises at least 3,000 different oligonucleotide members.

34. The method of claim 1, wherein the target nucleic acid, the first sequencing primer, the polymerase, the dNTPs, and the ddNTPs are mixed in a reservoir that is in fluid communication with a reagent introduction channel in the microfluidic device.

35. The method of claim 1, wherein the target nucleic acid, the first sequencing primer, the polymerase, the dNTPs, and the ddNTPs are mixed in a reagent mixing channel in the microfluidic device.

36. The method of claim 35, wherein the target nucleic acid, the first sequencing primer, the polymerase, the dNTPs, and the ddNTPs are provided in a plurality of different reservoirs that are in fluid communication with the reagent mixing channel, and are mixed in the microfluidic device by simultaneously transporting the target nucleic acid, the first sequencing primer, the polymerase, the dNTPs, and the ddNTPs into the reagent mixing channel.

37. The method of claim 34, wherein the reagent introduction channel intersects a separation channel, and the separating step comprises transporting the polymerization products through the reagent introduction channel into the separation channel, and separating the polymerization products.

38. The method of claim 35, wherein the reagent mixing channel intersects a separation channel, and the separating step comprises transporting the polymerization products from the reagent introduction channel into the separation channel, and separating the polymerization products.

39. The method of claim 37, wherein the polymerization products are separated in the separation channel by electrophoresis.

40. The method of claim 38, wherein the polymerization products are separated in the separation channel by applying a voltage differential along a length of the separation channel.

41. The method of claim 37, wherein the polymerization products are separated in the separation channel by electrophoresis.

42. The method of claim 37, wherein the polymerization products are separated in the separation channel by applying a voltage differential along a length of the separation channel.

43. The method of claims 37 or 38, wherein the polymerization products comprise fluorescent labels.

44. The method of claim 1, wherein the providing step comprises providing a plurality of sources of different sequencing primers that includes a source of a first sequencing primer.

45. The method of claim 44, wherein the plurality of sores of different sequencing primers are provided separate from the microfluidic device, and the mixing step comprises delivering the first sequencing primer from the plurality of sources of sequencing primer to the microfluidic device to mix with the polymerase the dNTPs, and the ddNTPs.

46. The method of claim 45, wherein the plurality of sources of sequencing primers comprises at least about 70% of all primer sequences having the length of the first primer sequence.

47. The method of claim 1, wherein the first sequencing primer comprises a plurality of separate primers that are capable of contiguously annealing to the target nucleic acid.

48. The method of claim 47, wherein each of the plurality of separate primers that makes up the first sequencing primer has a length of from about 4 to about 10 nucleotides.

49. The method of claim 1, wherein the first sequencing primer comprises at least three separate primers that are capable of contiguously annealing to the target nucleic acid.

50. The method of claim 47, wherein the plurality of separate primers that comprise the first sequencing primer are selected from a plurality of sources of primer sequences.

51. The method of claim 50, wherein each of the separate primer sequences in the plurality of sources of primer sequences has a length of from about 4 to about 10 nucleotides.

52. The method of claim 50, wherein the plurality of sources of primer sequences includes at least 70% of all possible primer sequences of a given length, wherein the given length is from about 4 to bout 10 nucleotides.

53. The method of claim 2, wherein the second sequencing primer comprises a plurality of separate primers that are capable of contiguously annealing to a portion of the first sequence of the target nucleic acid.

54. The method of claim 53, wherein each of the plurality of separate primers that makes up the second sequencing primer has a length of from about 4 to about 10 nucleotides.

55. The method of claim 2, wherein the second sequencing primer comprises at least three separate primers that are capable of contiguously annealing to a portion of the first sequence of the target nucleic acid.

56. The method of claim 55, wherein each of the separate primer sequences in the plurality of separate primer sequences has a length of from about 4 to about 10 nucleotides.

57. The method of claim 55, wherein the plurality of separate primer sequences are selected from a plurality of sources of primer sequences.

58. The method of claim 57, wherein the plurality of sources of primer sequences includes at least 70% of all possible primer sequences of a given length, wherein the given length is from about 4 to bout 10 nucleotides.

59. The method of claim 2, further comprising selecting a third primer sequence based upon the second sequence of the target nucleic acid, and mixing the third sequencing primer with the target nucleic acid under conditions to permit target dependent elongation of the third selected sequencing primer, thereby providing a third set of polymerization products which are separated by size in the microfluidic device to provide a third sequence of the target nucleic acid.

60. The method of claim 59, wherein the third sequencing primer comprises a plurality of separate primers that are capable of contiguously annealing to a portion of the second sequence of the target nucleic acid.

61. The method of claim 60, wherein each of the plurality of primers that makes up the third sequencing primer has a length of from about 4 to about 10 nucleotides.

62. The method of claim 61, wherein the third sequencing primer comprises at least three separate primers at are capable of contiguously annealing to a portion of the second sequence of target nucleic acid.

63. The method of claim 62, wherein each of the primer sequences in the plurality of primer sequences has a length of from about 4 to about 10 nucleotides.

64. The method of claim 63, wherein the plurality of separate primer sequences are selected from a plurality of sources of primer sequences.

65. The method of claim 64, wherein the plurality of sources of primer sequences includes at least 70% of all possible primer sequences of a given length, wherein the given length is from about 4 to bout 10 nucleotides.

6. A method of sequencing a target nucleic acid, comprising:

(a) providing an integrated microfluidic system comprising a microfluidic device comprising;

at least a first sequencing reaction channel and at least a first sequencing reagent introduction channel, the sequencing reaction channel and sequencing reagent introduction channel being in fluid communication; and

,

a fluidic interface in fluid communication with the sequencing reagent introduction channel for sampling a plurality of sequencing reagents or mixtures of sequencing reagents from a plurality of sources of sequencing reagents or mixtures of sequencing reagents and introducing the sequencing reagents or mixtures of sequencing reagents into the sequence reagent introduction channel from the sources of sequencing reagents or mixtures of sequencing reagents;

selecting a first sequencing primer sequence complementary to a first subsequence of a first target nucleic acid sequence;

(b) introducing the first sequencing primer and the first target nucleic acid sequence into the sequence reagent introduction channel;

(c) hybridizing the first primer sequence to the first subsequence in the first sequencing reaction channel and polymerase-extending the first primer sequence along the length of the target nucleic acid sequence to form a first extension product that is complementary to the first subsequence and a second subsequence of the target nucleic acid;

(d) determining the sequence of the first extension product;

(e) based upon the sequence of the first extension product, selecting a second primer sequence complementary to the second subsequence of the target nucleic acid sequence;

(f) hybridizing the second primer sequence to the second subsequence in the first sequencing reaction channel, or, optionally, hybridizing the second primer sequence in the second subsequence in a second sequencing reaction channel;

(g) extending the second primer sequence along the length of the target nucleic acid sequence to form a second extension product that is complementary to the second subsequence and a third subsequence of the target nucleic acid sequence; and

(h) determining the sequence of the second extension product.

7. The method of claim 6, wherein the second primer is selected using a computer.

8. The method of claim 6, wherein the microfluidic device comprises a material transport system for controllably transporting sequencing reagents through the sequencing reagent introduction channel and sequencing reaction channel.

9. A method of sequencing a nucleic acid comprising:

providing a set of sequencing primers, a target nucleic acid, a polymerase, dNTPs, and ddNTPs;

selecting a first primer from the set of primers;

introducing the first primer into a microfluidic device;

mixing the first primer, the polymerase, the dNTPs, and the ddNTPs in a first zone of the microfluidic device under conditions permitting target dependent polymerization of the dNTPs, thereby providing polymerization products;

separating polymerization products by size in a second zone of the microfluidic device to provide at least a first portion of the sequence of the target nucleic acid;

selecting a second primer from the set of primers, which primer is complementary to the first portion of the target nucleic acid;

introducing the second primer into the microfluidic device;

mixing the second primer, the polymerase, the dNTPs, and the ddNTPs in a third zone of the microfluidic device under conditions permitting target dependent polymerization of the dNTPs, thereby providing polymerization products;

separating polymerization products by size in a fourth zone of the microfluidic device to provide at least a second portion of the sequence of the target nucleic acid.

10. The method of claim 9, wherein the first and third zone of the microfluidic device are the same and wherein the second and fourth zone of the microfluidic device are the same.

11. The method of claim 9, wherein the set of primers is located on the microfluidic apparatus.

12. The method of claim 9, wherein mixing the second primer, polymerase, dNTPs, ddNTPs in a microfluidic device under conditions permitting polymerization and separating polymerization products by size to provide at least a portion of the sequence of the target nucleic acid is performed in less than 15 minutes.

13. The method of claim 9, further comprising selecting a third primer from the set of primers, which third primer is complementary to the second portion of the target nucleic acid;

mixing the third primer, the polymerase, the dNTPs, and the ddNTPs in a microfluidic device under conditions permitting target dependent polymerization of the dNTPs, thereby providing polymerization products;

separating polymerization products by size to provide at least a third portion of the sequence of the target nucleic acid.

14. An integrated method of performing a fluidic analysis of sample materials, comprising:

(a) providing an integrated microfluidic system comprising a microfluidic device comprising;

at least a first reaction chamber or channel, and at least a first reagent introduction channel, the first reaction chamber or channel and reagent introduction channel being in fluid communication;

a material transport system for controllably transporting a material through the reagent introduction channel and reaction chamber or channel;

a fluidic interface in fluid communication with the reagent introduction channel for sampling a plurality of reagents or mixtures of reagents from a plurality of sources of reagents or mixtures of reagents and introducing the reagents or mixtures of reagents into the reagent introduction channel;

(b) selecting a first set of DNA sequencing reagents comprising a first nucleic acid sequencing primer from the plurality of sources of sequencing reagents or mixtures of sequencing reagents;

(c) introducing a first DNA template and the first set of DNA sequencing reagents comprising a first nucleic acid sequencing primer into the first reaction chamber or channel whereupon the first DNA template and the first set of DNA sequencing reagents comprising a first nucleic acid sequencing primer react to produce a first set of DNA sequencing products;

(d) analyzing the first set of DNA sequencing products by separating the DNA sequencing products by size and detecting the size separated DNA sequencing products, thereby providing sequence information for the first DNA template;

(e) based upon the the sequence information determined in step (d), selecting a second set of DNA sequencing reagents comprising a second sequencing primer and a second DNA template;

(f) introducing the second set of DNA sequencing reagents comprising a second sequencing primer and the first DNA template into the first reaction chamber or channel, or, optionally, into a second reaction chamber or channel in the microfluidic device, whereupon the second set of DNA sequencing reagents comprising a second sequencing primer and the first DNA template react to produce a second set of DNA sequencing products; and

(g) analyzing the second set of DNA sequencing products to provide additional sequence information for the first DNA template.

15. The method of claim 14, wherein the first or second reagents or mixtures of reagents comprise a thermostable polymerase and the method further comprises heating the first sample material and the first reagent or mixture of reagents or the second sample materials and the second reagent or mixture of reagents.

16. The method of claim 14, wherein the second sequencing primer is selected using a computer.

17. A method of performing iterative fluid operations, comprising:

providing a microfluidic system which comprises;

a microfluidic device having at least a first microscale channel disposed therein, the at least first channel being in fluid communication with a plurality of reagent sources;

a material transport system for transporting reagents from each of the plurality of reagent sources into the at least first channel;

introducing at least a first nucleic acid template from at least a first reagent source and a first sequencing primer from at least a second reagent source into the at least first channel, the nucleic acid template and first sequencing primer producing a first elongation product;

detecting a nucleotide sequence of the first elongation product; and

based upon the reaction of the first and second reagents, selecting and introducing a an additional amount of the nucleic acid template and a second sequencing primer into the at least first channel the second sequencing primer based upon the nucleotide sequence of the first elongation product.

18. The method of claim 17, wherein the second sequencing primer comprises a sequence identical to at least a portion of the first elongation reaction product.

19. The method of claim 17, wherein the at least first sequencing primer comprises a plurality of nucleic acid primers that contiguously anneal to the nucleic acid template.

20. The method of claim 17, wherein the at least first sequencing primer comprises at least 2 contiguously annealing primers.

21. The method of claim 17, wherein the at least first sequencing primer comprises at least 3 contiguously annealing primers.

22. The method of claim 19, wherein the plurality of contiguously annealing primers are 5 or 6 nucleotides in length.

23. The method of claim 19, wherein the plurality of contiguously annealing primers are independently selected from a plurality of primer sources comprising all possible primers of 5 or 6 nucleotides in length.

24. The method of claim 17, wherein the second sequencing primer comprises a plurality of nucleic acid primers that contiguously anneal to the nucleic acid template.

25. The method of claim 17, wherein the at least second sequencing primer comprises at least 2 contiguously annealing primers.

26. The method of claim 17, wherein the at least second sequencing primer comprises at least 3 contiguously annealing primers.

27. The method of claim 24, wherein the plurality of contiguously annealing primers are 5 or 6 nucleotides in length.

28. The method of claim 24, wherein the plurality of contiguously annealing primers are independently selected from a plurality of primer sources comprising all possible primers of 5 or 6 nucleotides in length.

29. The method of claim 17, further comprising;

repeating at least 10 times, the steps of selecting and introducing additional sequencing primers to the at least first channel along with the template nucleic acid based upon the nucleotide sequence of a previous elongation reaction product, the additional sequencing primers reacting with the nucleic acid template to produce an additional elongation reaction product.

30. The method of claim 17, further comprising:

repeating at least 20 times, the steps of selecting and introducing additional sequencing primers to the at least first channel along with the template nucleic acid based upon the nucleotide sequence of a previous elongation reaction product, the additional sequencing primers reacting with the nucleic acid template to produce an additional elongation reaction product.

31. The method of claim 17, further comprising:

repeating at least 50 times, the steps of selecting and introducing additional sequencing primers to the at least first channel along with the template nucleic acid based upon the nucleotide sequence of a previous elongation reaction product, the additional sequencing primers reacting with the nucleic acid template to produce an additional elongation reaction product.

32. The method of claim 17, further comprising:

repeating at least 100 times, the steps of selecting and introducing additional sequencing primers to the at least first channel along with the template nucleic acid based upon the nucleotide sequence of a previous elongation reaction product, the additional sequencing primers reacting with the nucleic acid template to produce an additional elongation reaction product.

33. The method of claim 17, further comprising:

repeating the steps of selecting and introducing additional sequencing primers to the at least first channel along with the template nucleic acid based upon the nucleotide sequence of a previous elongation reaction product, the additional sequencing primers reacting with the nucleic acid template to produce an additional elongation reaction product, until a nucleotide sequence of substantially all of the template nucleic acid is determined.