THERMAL CYCLING SYSTEM

• US 20080003649A1
• Filed: 05/17/2007
• Published: 01/03/2008
• Est. Priority Date: 05/17/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Walker, Christopher, Goodwin, David, Scherer, Axel, Maltezos, George, Johnston, Matthew
• Granted Patent: US 8,232,091 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/185 using a liquid as fluid
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/50853 with covers or lids
• 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: B01L 7/54 using spatial temperature gradients
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters

Abstract

This invention provides a system for performing PCR, and real time PCR in particular with great speed and specificity. The system employs a heat block containing a liquid composition to rapidly transfer heat to and from reaction vessels. The system makes use of the reflective properties of the liquid metal to reflect signal from the PCR into the vessel and out the top. In this way, the signal can be measured by an optical assembly in real time without removing the vessels from the heat block.

Claims

1. A method of performing PCR comprising:

a) performing PCR in a sample vessel and generating a signal in the sample vessel indicating the course of the PCR, wherein substantially all said signal is reflected back into said sample vessel, wherein substantially all the signal is detected from said sample vessel from a discrete location; and

b) measuring the signal emitted from the discrete location of the sample vessel.

2. The method of claim 1 comprising measuring the signal over a plurality of PCR cycles in real time.

3. The method of claim 2 wherein the sample vessel comprises a wall transparent to the signal and at least part of the wall is immersed in a liquid composition that reflects substantially all the signal striking the liquid composition.

4. The method of claim 3 wherein the liquid composition comprises a metal or metal alloy.

5. The method of claim 3 wherein the signal is generated by a label or dye.

6. The method of claim 1 wherein said sample vessel comprises a material that reflects substantially all said signal in said sample vessel.

7. The method of claim 1, wherein said sample vessel is comprised of a metal.

8. The method of claim 1, wherein said sample vessel comprises a reflective material.

9. The method of claim 2, wherein said sample vessel is separated from said liquid composition by a receptacle.

10. A method of performing PCR comprising:

a) performing PCR of a target nucleotide sequence in a reaction mixture under conditions wherein;

(i) the rate of temperature change between primer extension and duplex dissociation and between primer annealing and primer extension is more than 10.5°

C. per second

(ii) said PCR is conducted in a heat block comprising sample vessels, comprising temperature variance of less than 0.5°

C. within a sample vessel; and

b) monitoring amplification of the target nucleotide sequence in real time.

11. The method of claim 10, wherein said heat block comprises a liquid composition.

12. The method of claim 10, further comprising a temperature variance of less than 0.5°

C. as measured between two or more wells in said heat block.

13. The method of claim 12, wherein said temperature variance is 0.01°

C. as measured between two or more wells in said heat block.

14. The method of claim 10, wherein said heat block is a swap block.

15. The method of claim 10 wherein the rate of temperature change is effected by:

(1) putting a sample vessel containing the reaction mixture in thermal contact with a liquid composition having a heat transfer coefficient of at least 0.1 W/M*K;

wherein the temperature of the liquid composition controls the temperature of the reaction mixture.

16. The method of claim 15, comprising maintaining temperature uniformity by inducing movement of the liquid composition.

17. A method of performing PCR comprising:

conducting PCR in a thermal cycler which modulates sample temperature by more than 5°

C. per second;

wherein said thermal cycler temperature is regulated by a liquid composition in heat block with a liquid tight seal;

wherein said liquid composition comprises a heat transfer coefficient of at least 0.1 W/M*K.

18. The method of claim 17 wherein said thermal cycler provides ramp rates of at least about 10°

C. per second.

19. The method of claim 17, wherein said thermal cycler provides well-to-well and sample temperature uniformity of at least about 0.01 to 0.1°

C.

20. The method of claim 18, wherein said heat block has a temperature variance is 0.01°

C. as measured between two or more wells in said heat block.

21. The method of claim 18, wherein said liquid composition is contained within a heat block.

22. The method of claim 20, wherein said heat block is a swap block.

23. A method of performing PCR comprising;

conducting PCR in a thermal cycler which modulates sample temperature sufficiently to allow detecting amplification in real time at a signal index of at least 3, wherein said detection is via a non-specific nucleic acid label, wherein said thermal cycler modulates sample temperature by more than 10°

C. per second.

24. A method for performing real-time PCR comprising:

a) performing a PCR reaction of a target nucleotide sequence in a reaction mixture under conditions wherein the rate of temperature change between primer extension and duplex dissociation and between primer annealing and primer extension in the reaction mixture is at least 15°

C. per second, wherein said PCR reaction is in thermal contact with a liquid composition having heat transfer coefficient of at least 0.1 W/m*K; and

b) monitoring the PCR in real time.

25. The method of claim Error! Reference source not found, wherein monitoring comprises:

a) performing PCR in a sample vessel and generating signal in the sample vessel indicating the course of the PCR, wherein the signal is emitted from the sample vessel substantially from a discrete location; and

b) measuring the signal emitted from the discrete location of the sample vessel.

26. The method of claim Error! Reference source not found wherein the rate of temperature increase in the reaction mixture is at least 40°

per second.

27. The method of claim Error! Reference source not found wherein said temperature change is regulated by a Peltier element.

28. A method for performing real-time PCR comprising:

a) cycling the temperature of the PCR reaction mixture between temperatures for duplex dissociation, primer annealing and primer extension for a plurality of cycles, wherein each cycle is no more than five seconds;

wherein said temperatures are modulated by a liquid composition sealed in a thermal cycler;

b) monitoring the course of PCR in the sample vessel in real time over a plurality of cycles.

29. The method of claim 28 further comprising before step (a):

i) placing a closed end of a sample vessel containing the PCR reaction mixture into thermal contact with a liquid composition that is liquid above 60°

C. and that has a heat transfer coefficient of at least 0.1 watts/meter-degree Kelvin, whereby the temperature of the liquid composition controls the temperature of the PCR reaction mixture.

30. The method of claim 28 wherein the liquid composition reflects substantially all light inside the sample vessel.

31. The method of claim 30 wherein monitoring is performed by measuring signal emitted from the top or bottom of the sample vessel.

32. The method of claim 28 wherein each cycle is no more than three seconds.

33. The method of claim 29, wherein said sample vessel comprises a reflective surface.

34. The method of claim 29, wherein said sample vessel is further covered with a cap.

35. The method of claim 34, wherein said cap is capable of absorbing heat from or being cooled by said liquid composition.

36. A method for conducting real time PCR comprising providing a thermal cycler comprising a liquid composition in thermal contact with PCR sample vessels;

wherein said liquid composition modulates in temperature thus regulating reaction temperatures in the sample vessels, wherein a detectable signal is emitted from said sample vessels; and

detecting said signal via an optical assembly comprising a light emitter and optical detector.

37. The method of claim 36, wherein said optical assembly comprises a pin photodiode CCD imager, a CMOS imager, a line scanner, a photodiode, a phototransistor, a photomultiplier or an avalanche photodiode.

38. The method of claim 36, wherein said sample vessel is further covered with a cap.

39. The method of claim 39, wherein said cap is capable of absorbing heat from or being cooled by said liquid composition.

40. An apparatus comprising:

a) a temperature control assembly comprising a container containing a liquid composition that is liquid above 60°

C. and that has a thermal conductivity of at least 0.1 watts/meter-degree Kelvin, said container having at least one aperture, each aperture adapted to receive a closed end of an sample vessel, wherein a sample vessel received into the aperture is placed in thermal contact with the liquid composition whereby the temperature of the liquid composition controls the temperature of a liquid sample in the sample vessel and wherein the liquid composition is capable of reflecting substantially all light inside the sample vessel;

b) an optical assembly capable of detecting said light inside the sample vessel from a discrete location on said sample vessel; and

c) a control assembly that controls the temperature of the liquid composition and the operation of the optical assembly.

41. The apparatus of claim 40 wherein the liquid composition comprises gallium a gallium-indium alloy or alloy comprising gallium, indium, rhodium, silver, zinc, tin or stannous.

42. The apparatus of claim 40 wherein temperature controller comprises a Peltier element.

43. The apparatus of claim 40 wherein temperature controller comprises resistive wire in thermal contact with the liquid composition.

44. The apparatus of claim 40 wherein temperature controller comprises means to cycle the temperature of the liquid composition between temperatures for duplex dissociation, primer annealing and primer extension appropriate for PCR.

45. The apparatus of claim 40 wherein thermal cycler further comprises means for circulating current in the liquid composition.

46. The apparatus of claim 40 wherein the optical assembly comprises a light emitter and optical detector.

47. The apparatus of claim 40 further comprising:

a sample preparation station comprising means to add reagents to sample vessels.

48. The apparatus of claim 40 further comprising means for moving the sample vessels into the apertures.

49. The apparatus of claim 47 further comprising a digital computer that controls the thermal cycler, the optical assembly and the sample preparation station.

50. A system for performing real time PCR comprising:

a) a thermal cycler comprising a liquid composition and means to engage a sample vessel and put said vessel in thermal contact with the composition; and

b) an optical assembly comprising a light emitter and optical detector that directs light into an engaged sample vessel and detects light emitted from an engaged sample vessel.

51. The system of claim 50, wherein said liquid composition comprises a metal or metal alloy.

52. The system of claim 50, wherein said liquid composition comprises gallium.

53. The system of claim 50, wherein said signals emitted are from a removable cap of said sample vessel.

54. The system of claim 50, wherein said liquid composition is separated from said sample vessel by a receptacle.

55. The system of claim 54, wherein said receptacle is transparent or transluscent.

56. The system of claim 50, wherein said system comprises a Peltier element.

57. The system of claim 56, wherein said thermal cycler further comprises a motor operatively connected to a fan and stir bar.

58. The system of claim 57, wherein said fan and stir bar are connected coaxially to said motor.

59. The system of claim 50, wherein said thermal cycler further comprises a resistive wire for thermal regulation of said liquid composition.

60. The system of claim 50, wherein said liquid composition is sealed in a closed barrier, wherein said barrier comprises a surface with receptacles into which said sample vessels are placed.

61. The system of claim 50, wherein said liquid composition directly contacts said sample vessels.

62. The system of claim 50 wherein said optical assembly comprises a PIN photodiode, CCD imager, a CMOS imager, a line scanner, a photodiode, a phototransistor, a photomultiplier or an avalanche photodiode.

76. The apparatus of claim 61 wherein said first piece or second piece comprises a reflective surface.

63. An apparatus comprising:

a) a heat sink;

b) a heating component in thermal contact with the heat sink;

c) a barrier comprising a wall having a top and bottom surfaces, wherein the bottom surface is sealed to the said heating component wherein the sealed barrier and said heating component form a container containing a liquid composition;

d) a first piece comprising a plurality of wells, wherein the first piece is sealed to the top surface of the barrier and the wells extend into the container;

e) a second piece comprising a plurality of sample vessels, each with an open end, wherein the sample vessels are removably inserted into the wells;

f) a third piece comprising a plurality of extrusions, wherein the extrusions are removably inserted into the open ends of the sample vessels.

64. The apparatus of claim 63, wherein said liquid composition comprises a metal that is liquid above 60°

C. and that has a heat transfer coefficient of at least 0.1 M/W/K.

65. The apparatus of claim 63, wherein said liquid metal is capable of increasing in volume by about 0.1 to 6.0%.

66. The apparatus of claim 63, wherein said capable of an increase in temperatures is more than 10.5°

C./second.

67. The apparatus of claim 63, wherein said well in e) is adapted to be flexible so that said well is capable of forming a thermal or optical contact with said sample vessels in f).

68. The apparatus of claim 67, wherein said adapting comprises utilizing a geometric configuration and/or deformable material in said well.

69. The apparatus of claim 68, wherein said geometric configuration is polygonal, elliptical or circular.

70. The apparatus of claim 63 wherein said wells are immersed in said liquid composition to an initial level.

71. The apparatus of claim 70, wherein said extrusion extends to above, at or below said initial level.

72. The apparatus of claim 63, wherein said third piece is capable of absorbing heat from or being cooled by said liquid composition.

73. The apparatus of claim 63 wherein the wells of the first piece are transparent and flexible, and the sample vessels of the second piece are transparent, whereby increased pressure on said wells places said liquid in said container in thermal and optical contact with a sample in said sample vessel.

74. The apparatus of claim 63 wherein the extrusions of the third piece are transparent.

75. The apparatus of claim 63 wherein the barrier is sealed to each of a heat spreader and the first piece through a gasket.

77. The apparatus of claim 63 wherein the barrier is sealed to each of a heat spreader and the first piece with a fastener.

78. The apparatus of claim 63 further comprising a heat spreader in contact with said heating component.

79. The apparatus of claim 63 wherein said heating component is a Peltier, wire elements or a combination thereof.

80. The apparatus of claim 63 further comprising a second heating component, wherein said barrier is disposed between said heating component and said second heating component.

81. The apparatus of claim 63 or claim 69 wherein said plurality of wells comprise 4, 8, 16, 32, 48, 96, 196, 384 or 1536 wells.

82. The apparatus of claim 63 or claim 69, farther comprising a fan and stir bar, wherein optionally said fan and stir bar are operatively connected to a single motor.

83. The apparatus of claim 82, wherein said apparatus is powered by a battery.

84. The apparatus of claim 82, wherein said fan is turned on and off automatically by a controller component operatively connected to said apparatus.

85. The apparatus of claim 63, further comprising a metal heat spreader.

86. The apparatus of claim 85, wherein said spreader comprises copper.

87. The apparatus of claim 63, wherein said heating component is configured for gradient PCR.

88. A continuous flow PCR system comprising a sample preparation module;

thermal cycler, wherein said thermal cycler comprises a liquid composition for modulating temperature in said sample; and

an optical assembly for detecting an emission signal from said sample.

89. The system of claim 88, further comprising a sampler and a waste collection.