Methods for rapid multiplexed amplification of target nucleic acids

• US 20090023603A1
• Filed: 04/04/2008
• Published: 01/22/2009
• Est. Priority Date: 04/04/2007
• Status: Active Grant

• Current Assignee: ANDE Corp.
• Original Assignee: Network Biosystems, Inc.
• Inventors: Giese, Heidi Susanne, Tan, Eugene, Selden, Richard F., Lam, Heung Chuan, Wright, John A., Kellogg, Gregory John
• Granted Patent: US 8,425,861 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• 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 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• 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 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: G01N 2021/6441 : with two or more labels
• CPC Class Codes: G01N 21/6402 : Atomic fluorescence; Laser induced fluorescence
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6486 : Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 27/44726 : using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G01N 27/44782 : of a plurality of samples
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A fast, multiplexed PCR system is described that can rapidly generate amplified nucleic acid products, for example, a full STR profile, from a target nucleic acid. Such systems include, for example, microfluidic biochips and a custom built thermal cycler, which are also described. The resulting STR profiles can satisfy forensic guidelines for signal strength, inter-loci peak height balance, heterozygous peak height ratio, incomplete non-template nucleotide addition, and stutter.

Claims

1. A thermal cycler comprising:

a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor,

wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature.

48. The method of claim 45, wherein the thermal cycling is provided by a thermal cycler of claim 1.

2. A thermal cycler comprising:

a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a first thermosensor,

wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature, further comprising a second thermosensor positioned to monitor the temperature of said first surface of said TCE.

4. The thermal cycler of claim 2, further comprising a chip compression element (CCE) positioned over the first surface of the TCE allowing insertion of a substrate between the CCE and the TCE, wherein the CCE provides for thermal communication between the TCE and the substrate.

5. The thermal cycler of claim 4, wherein the CCE comprises a low thermal mass and insulating material.

6. The thermal cycler of claim 4, wherein the CCE is a member of the group consisting of a foam pad, one or a plurality of clips and an air bladder.

7. The thermal cycler of claim 2 wherein the first surface of the TCE is adapted to receive a thin-walled tube.

8. The thermal cycler of claim 2 having a heating or cooling rate at the first surface of the TCE of about 4-150°

C. per second.

9. The thermal cycler of claim 2, further comprising a heat sink comprising a variable speed cooling fan for controlling the temperature of the heat sink.

10. The thermal cycler of claim 9, wherein the heat sink further comprises a second thermocycler for controlling the temperature of the heat sink.

11. The thermal cycler of claim 2, having a temperature stability of about +/−

1.0°

C. at a sample in a substrate in thermal communication with the first surface of the TCE.

12. The thermal cycler of claim 2, wherein the TCE comprises a high heating and cooling capacity heat pump or a high power output Peltier device.

13. The thermal cycler of claim 9, wherein the heat sink is a fan-cooled heat sink with copper bases and cooling fins.

14. The thermal cycler of claim 13, wherein the heat sink has a thermal resistance of approximately 0.4°

C./W or less.

3. A thermal cycler comprising:

a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber, said chamber containing a solution and a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature.

15. A system comprising

a biochip comprising one or a plurality of reaction chambers, wherein each reaction chamber comprises a microfluidic inlet channel and a microfluidic outlet channel, wherein each reaction chamber is less than 200 μ

m from a contact surface of the biochip substrate;

and a thermal cycler, comprising

a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature,

in thermal communication with the contact surface of the biochip substrate.

16. A system comprising

a biochip comprising one or a plurality of reaction chambers, wherein each reaction chamber comprises a microfluidic inlet channel and a microfluidic outlet channel, wherein each reaction chamber is less than 100 μ

m from a contact surface of the biochip substrate;

and a thermal cycler, comprising

a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature, further comprising a second thermosensor positioned to monitor the temperature of said first surface of said TCE,

in thermal communication with the contact surface of the biochip substrate.

17. The system of claim 16 wherein the microfluidic inlet channel, the microfluidic outlet channel, or both has a via.

18. The system of claim 16, wherein the thermal cycler further comprises a chip compression element (CCE) positioned over the first surface of the TCE allowing insertion of a substrate between the CCE and the TCE, wherein the CCE provides for thermal communication between the TCE and the substrate.

19. The system of claim 16, wherein the chip compression element comprises a low thermal mass and insulating material.

20. The system of claim 18, wherein the CCE is selected from the group consisting of a foam pad, one or a plurality of clips and an air bladder.

21. The system of claim 18, wherein the CCE provides about 5 to about 50 psi of pressure to hold the contact surface of the biochip substrate in thermal contact with the first surface of the TCE.

22. The system of claim 16, wherein the reaction chambers are not coated with a polymer or silane coating or BSA.

23. The system of claim 16, wherein thermal communication between the contact surface of the biochip substrate and the first surface of the TCE is provided in the absence of a thermal coupling solution.

24. The system of claim 16, having a heating or cooling rate at the first surface of the TCE of about 4-150°

C. per second.

25. The system of claim 16, wherein the thermal cycler is capable of a heating or cooling rate within the reaction chambers of about 4-150°

C. per second.

26. The system of claim 16, further comprising a fan-cooled heat sink with copper bases and cooling fins.

27. The system of claim 16, further comprising a heat sink with a thermal resistance of approximately 0.4°

C./W or less.

28. The system of claim 16, further comprising a heat sink with a variable speed cooling fan for controlling the temperature of the heat sink.

29. The system of claim 16, further comprising a heat sink further comprising a second heating element for controlling the temperature of the heat sink.

30. The system of claim 16, wherein the thermal cycler is capable of a temperature stability of +/−

1.0°

C. at a sample in the biochip.

31. The system of claim 16, wherein the TCE comprises a high heating and cooling capacity heat pump or a high power output Peltier device.

32. The system of claim 16, wherein the biochip substrate is constructed of an organic material, an inorganic material, a crystalline material or an amorphous material.

33. The system of claim 16, wherein the biochip substrate comprises a plastic material.

34. The system of claim 33, wherein the biochip substrate comprises a cyclic olefin co-polymer (COC).

35. The system of claim 16, wherein the biochip substrate comprises 8-128 microfluidic systems.

36. The system of claim 16, wherein each reaction chamber has a volume of less than about 100 μ

L.

37. A method for simultaneously amplifying of a plurality of loci in a nucleic acid solution comprising

providing one or a plurality of reaction chambers wherein

each reaction chamber comprises

(i) a nucleic acid solution comprising at least one copy of at least one target nucleic acid to be amplified;

(ii) one or more buffers;

(iii) one or more salts;

(iv) a primer set corresponding to each of the plurality of loci to be amplified;

(v) a nucleic acid polymerase; and

(vi) nucleotides,

sequentially thermally cycling the temperature of the nucleic acid solution in each reaction chamber between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles at heating and a cooling rates of about 4-150°

C./sec, to yield a plurality of amplified loci in each reaction chamber in about 97 minutes or less.

38. The method of claim 37, further comprising

holding the one or a plurality of reaction solutions at a final state to provide one or a plurality of amplified nucleic acid products.

39. A method for simultaneously amplifying of a plurality of loci in a nucleic acid solution comprising

providing one or a plurality of reaction chambers wherein

each reaction chamber comprises

(i) a nucleic acid solution comprising at least one copy of at least one target nucleic acid to be amplified;

(ii) one or more buffers;

(iii) one or more salts;

(iv) a primer set corresponding to each of the plurality of loci to be amplified;

(v) a nucleic acid polymerase; and

(vi) nucleotides,

sequentially thermally cycling the temperature of the nucleic acid solution in each reaction chamber for a predetermined number of cycles at heating and a cooling rates of about 4-150°

C./sec, to yield a plurality of amplified loci in each reaction chamber in about 97 minutes or less.

40. A method for simultaneously amplifying 5 or more loci in a nucleic acid solution comprising

providing one or a plurality of reaction chambers wherein

each reaction chamber comprises.

(i) a nucleic acid solution comprising at least one copy of at least one target nucleic acid to be amplified;

(ii) one or more buffers;

(iii) one or more salts;

(iv) a primer set corresponding to the 5 or more loci to be amplified;

(v) a nucleic acid polymerase; and

(vi) nucleotides,

sequentially thermally cycling the temperature of the nucleic acid solution in each reaction chamber between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles at heating and a cooling rates of about 4-150°

C./sec, to yield 5 or more amplified loci in each reaction chamber.

41. The method of claim 40 wherein said nucleic acid solution is present in a biochip.

42. The method of claim 40 wherein said nucleic acid solution is present in a thin walled tube.

43. The method of claim 40, wherein the 5 or more amplified loci are produced in less than about 97 minutes.

44. The method of claim 40, wherein the amplified loci are produced in less than about 45 minutes.

45. The method of claim 40 further comprising, prior to the sequential thermal cycling,

heating the one or a plurality of reaction solutions to a first temperature suitable for hot-start activation of the nucleic acid polymerases;

and

holding the one or a plurality of reaction solutions at the first temperature for a first period of time suitable for hot-start activation of the nucleic acid polymerases.

46. The method of claim 45, wherein the first period of time is less than about 90 seconds.

47. The method of claim 45 wherein the first temperature is about 90 to about 99°

C.

49. The method claim 44, wherein the nucleic acid polymerase has an extension rate of at least 100 bp/sec.

50. The method of any one of claims 44, wherein each reaction chamber has a volume of less than about 100 μ

L.

51. The method of any one of claims 44, wherein each reaction chamber is separated from a thermal cycler by less than about 200 μ

m.

52. The method of any one of claims 44, wherein each nucleic acid solution comprises about 1 to about 1000 copies of a target nucleic acid.

53. The method claim 44, wherein the nucleic acid polymerase is SpeedSTAR, PHUSION, Hot MasterTaq™

, PHUSION Mpx, PyroStart, KOD, Z-Taq, or CS3AC/LA.

54. The method of claim 44, wherein analysis of each of the amplified nucleic acid products satisfies forensic interpretation guidelines.

55. The method of claim 44, wherein the denaturing state is about 95°

C. for about 4 seconds.

56. The method of claim 44, wherein the annealing state is about 59°

C. for about 15 seconds.

57. The method of claim 44, wherein the extension state is about 72°

C. for about 7 seconds.

58. The method claims 44, wherein the final state is about 70°

C. for about 90 seconds.

59. The method claims 44, wherein the one or a plurality of reaction solutions are cooled from the denaturing state to the annealing state at a first cooling rate of about 10 to about 50°

C./sec.

60. The method of claim 44, wherein the one or a plurality of reaction solutions are heated from the annealing state to the extension state at a first heating rate of about 10 to about 50°

C./sec.

61. The method claim 44, wherein the one or a plurality of reaction solutions are heated from the extension state to the denaturing state at a second heating rate of about 10 to about 50°

C./sec.

62. The method of claim 44, wherein the one or a plurality of amplified nucleic acid products are obtained in about 10 to about 90 minutes.

63. The method of claim 44, wherein each reaction solution comprises about 0.005 to about 10 ng of a target nucleic acid.

64. The method of claim 44, wherein the target nucleic acid comprises a human nucleic acid, microbial nucleic acid, or viral nucleic acid.

65. The method of claim 44, wherein 10 to 250 loci are simultaneously amplified.

66. The method of claim 65, wherein the loci comprise amelogenin, D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, or a plurality thereof.

67. The method of claim 44, wherein the predetermined number of cycles is between about 10 and about 50 cycles.

68. The method of claim 44, wherein one or a plurality of thin-wall reaction tubes comprise the one or a plurality of reaction chambers.

69. An integrated biochip system comprising

a biochip comprising at least two reaction chambers in microfludic communication, wherein a first reaction chamber is in thermal communication with a thermal cycler, comprising:

a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature,

wherein a contact surface of the biochip is in thermal communication with the first surface of the thermal cycler;

and

a second reaction chamber in fluid connection with the first reaction chamber and adapted for

(i) nucleic acid extraction;

(ii) nucleic acid purification;

(iii) pre-PCR nucleic acid cleanup;

(iv) post-PCR cleanup;

(v) pre-sequencing cleanup;

(vi) sequencing;

(vii) post-sequencing cleanup;

(viii) nucleic acid separation;

(ix) nucleic acid detection;

(x) reverse transcription;

(xi) pre-reverse transcription cleanup;

(xii) post-reverse transcription cleanup;

(xiii) nucleic acid ligation;

(xiv) nucleic acid hybridization;

(xv) quantification

wherein the first reaction chamber is less than 200 μ

m from a contact surface of the biochip.