APPARATUS FOR THERMAL CYCLING

• US 20120171678A1
• Filed: 03/09/2012
• Published: 07/05/2012
• Est. Priority Date: 05/17/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: MALTEZOS, George, JOHNSTON, Matthew, GOODWIN, David, SCHERER, Axel, WALKER, Christopher I.
• Granted Patent: US 9,316,586 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 thermal cycler apparatus comprising:

a heat sink;

a heating component in thermal contact with the heat sink;

a closed barrier sealing in a liquid composition, the closed barrier having a top surface and a bottom surface, the top surface having a plurality of wells configured to receive a plurality of sample vessels, and wherein the bottom surface is in thermal contact with the heating component; and

a control assembly in communication with the heating component, the control assembly configured to cycle the temperature of samples in the plurality of sample vessels for nucleic acid amplification.

2. The apparatus of claim 1, wherein the bottom surface comprises a metal base plate.

3. The apparatus of claim 1, further comprising a sample plate including the plurality of sample vessels, each of the plurality of sample vessels having an open end, and the sample plate configured to be removably inserted into the plurality of wells.

4. The apparatus of claim 3, wherein at least one sample vessel comprises a reflective surface.

5. The apparatus of claim 3, further comprising a cap plate configured to cover the open ends of the plurality of sample vessels.

6. The apparatus of claim 5, wherein the cap plate comprises a plurality of extrusions removably inserted into the open ends of the sample vessels.

7. The apparatus of claim 6, wherein the plurality of extrusions comprise a light guide.

8. The apparatus of claim 1, wherein the liquid composition has a heat transfer coefficient of less than or equal to 8.0 W/m °

K.

9. The apparatus of claim 1, wherein the liquid composition has a heat transfer coefficient of less than or equal to 4.0 W/m °

K.

10. The apparatus of claim 1, further comprising at least one stirring element configured to actively mix the liquid composition.

11. The apparatus of claim 1, further comprising an optical assembly comprising a light emitter configured to direct light into the plurality of wells and an optical detector configured to detect light emitted from the samples.

12. The apparatus of claim 11, wherein the optical assembly further comprises:

an excitation filter;

a barrier filter; and

a dichroic mirror configured to direct light from the light emitter passed through the excitation filter to the sample vessels and to direct light emitted from the samples to the optical detector via the barrier filter.

13. The apparatus of claim 11, wherein the optical assembly comprises a plurality of light emitters configured to emit light at different wavelengths.

14. The apparatus of claim 1, wherein the control assembly is configured to cycle the temperature of the samples such that the temperature between samples in any two sample vessels positioned in the plurality of wells has a variance of not more than +/−

0.1°

C.

15. The apparatus of claim 1, wherein the control assembly is configured to cycle the temperature of the samples such that the temperature of the samples has a variance from a desired temperature of not more than +/−

0.1°

C.

16. The apparatus of claim 1, wherein the control assembly is configured to cycle the temperature of the samples in sample vessels positioned in the plurality of wells such that the temperature of the samples remains at a temperature of at least 30°

C. during nucleic acid amplification.

17. The apparatus of claim 1, wherein the heating component comprises a Peltier device.

18. The apparatus of claim 1, wherein control assembly is configured to cycle the temperature such that the heat block has a ramp rate of at least 5.0°

C. per second.

19. A thermal cycler apparatus comprising:

a heat block having a first surface comprising wells configured to receive sample vessels and a second surface opposite the first surface;

a container sealing in a liquid; and

means for controlling the temperature of samples in the sample vessels for nucleic acid amplification, the means for controlling temperature comprising a heating component in thermal contact with the liquid, wherein the heating component is in contact with the second surface of the heat block.

20. The apparatus of claim 19, wherein the liquid has a heat transfer coefficient of less than or equal to 6.0 W/m °

K.

21. The apparatus of claim 19, wherein the liquid has a heat transfer coefficient of less than or equal to 3.0 W/m °

K.

22. The apparatus of claim 19, further comprising stirring means configured to mix the liquid.

23. The apparatus of claim 19, further comprising an optical assembly wherein the optical assembly comprises a light emitter configured to direct light into the wells and an optical detector configured to detect light emitted from the samples.

24. The apparatus of claim 23, wherein the optical assembly further comprises:

an excitation filter;

a barrier filter; and

a dichroic mirror configured to direct light from the light emitter passed through the excitation filter to the sample vessels and to direct light emitted from the samples to the optical detector via the barrier filter.

25. The apparatus of claim 19, wherein the means for controlling temperature is configured to cycle the temperature of the samples such that the temperature between samples in any two sample vessels positioned in the wells has a variance of not more than +/−

0.1°

C.

26. A method of performing nucleic acid amplifications, the method comprising:

mixing a liquid in a sealed container, the liquid having a heat transfer coefficient of less than or equal to 6.0 W/m °

K;

cycling the temperature of a reaction mixture in sample vessels to perform multiple amplification cycles, wherein the reaction mixture comprises a polynucleotide sample, reagents for carrying out nucleic acid amplification, and a dye for detecting amplification, and wherein the sample vessels are each in thermal contact with the liquid in the sealed container;

regulating the temperature of the reaction mixture in each of the sample vessels using a heating component in thermal contact with the liquid; and

optically measuring the dyes between or during each of the multiple amplification cycles to determine a level of amplification while the sample vessels remain in thermal contact with the liquid.

27. The method of claim 26, wherein the temperature between the reaction mixture in any two sample vessels has a variance of less than +/−

0.2°

C.

28. The method of claim 26, wherein the nucleic acid amplification comprises a real time polymerase chain reaction.

29. The method of claim 26, wherein mixing the liquid is performed with at least one stir bar.

30. The method of claim 26, wherein the dye for detecting the level of amplification comprises a fluorescent DNA-binding dye.

31. The method of claim 26, wherein the dye for detecting the level of amplification comprises a FRET-based probe.

32. The method of claim 26, wherein the nucleic acid amplification comprises a series of up to 30 polymerase chain reaction cycles completed in 1 to 20 minutes.