Thermal cycling by positioning relative to fixed-temperature heat source

• US 20100075296A1
• Filed: 07/29/2009
• Published: 03/25/2010
• Est. Priority Date: 07/31/2008
• Status: Active Grant

• Current Assignee: Genomadix Incorporated
• Original Assignee: Spartan Bioscience, Inc.
• Inventors: Shayanpour, Alan, Harder, Chris, Cloake, Martin, Perreault, Michael, Lem, Paul
• Granted Patent: US 8,945,880 B2
• Time in Patent Office: Days
• US Class Current: 435/3
• CPC Class Codes: B01L 2200/142 Preventing evaporation
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/042 Caps; Plugs
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 3/5082 Test tubes per se
• 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 9/06 Test-tube stands; Test-tube holders

Abstract

A thermal cycling system and method are provided herein, The thermal cycling system for performing a biological reaction at two or more different temperatures comprises: a) a heat source for setting at a fixed temperature; b) a reaction vessel containing material upon which the biological reaction is to be performed; c) mechanically-operable means for altering the relative position of the heat source and the reaction vessel so that reaction vessel first achieves and maintains a desired first temperature in the reaction vessel for starting the carrying out of the biological reaction, and then for altering the relative position of the heat source and the reaction vessel so that reaction vessel then achieves and maintains a second temperate for continuing the carrying out of the biological reaction on the biological material, and d) temperature-sensing means operatively associated with the reaction vessel for controlling the altering of the relative position of the heat source and the reaction vessel so that the reaction vessel achieves and maintains the desired second temperature in the reaction vessel.

Claims

1. A thermal cycling system for performing a biological reaction at two or more different temperatures:

the thermal cycling system comprising;

a) a heat source for setting at a fixed temperature;

b) a reaction vessel containing material upon which the biological reaction is to be performed;

c) mechanically-operable means for altering the relative position of the heat source and the reaction vessel so that reaction vessel first achieves and maintains a desired first temperature in the reaction vessel for starting the carrying out of the biological reaction, and then for altering the relative position of the heat source and the reaction vessel so that reaction vessel then achieves and maintains a second temperate for continuing the carrying out of the biological reaction on the biological material, and d) temperature-sensing means operatively associated with the reaction vessel for controlling the altering of the relative position of the heat source and the reaction vessel so that the reaction vessel achieves and maintains the desired second temperature in the reaction vessel.

3. The thermal cycling system, of claim 1, wherein said heat source is a block of heat retentive material including means to heat said block to, and maintain said block at a fixed temperature.

4. The thermal cycling system of claim 3, wherein said block is configured and arranged to be movable.

5. The thermal cycling system of claim 3, wherein said reaction vessel is embedded in a metal sleeve, and wherein said metal sleeve is configured and arranged to be movable.

6. The thermal cycling system of claim 5, wherein said sleeve includes a temperature sensor.

7. The thermal cycling system of claim 6 wherein said temperature sensor, upon sensing that the temperature of said sleeve approaches the desired denaturation temperature, instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired denaturation temperature.

8. The thermal cycling system of claim 6, wherein said temperature sensor, upon sensing that the temperature of said sleeve approaches the desired primer annealing temperature, instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired primer annealing temperature.

9. The thermal cycling system of claim 6, wherein said temperature sensor, upon sensing that the temperature of said sleeve approaches the desired primer extension temperature, instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired primer extension temperature.

10. The thermal cycling system of claim 5, wherein said temperature sensor in said sleeve is operatively associated with a processor which is downloaded with an algorithm to predict the temperature being experienced by said reaction vessel, said algorithm being based on a program to achieve and maintain a desired temperature in the reaction vessel.

11. The thermal cycling system of claim 10, wherein, when said temperature sensor in said sleeve which is operatively associated with said algorithm, senses that the temperature approaches the desired template denaturation temperature, instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired template denaturation temperature.

12. The thermal cycling system of claim 10, wherein, when said temperature sensor in the sleeve which is operatively associated with said algorithm, senses that the temperature approaches the desired primer annealing temperature, instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired primer annealing temperature.

13. The thermal cycling system of claim 10, wherein, when said temperature sensor in said sleeve which is operatively associated with said algorithm, senses that the temperature approaches the desired primer extension temperature, instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired primer extension temperature.

14. The thermal cycling system of claim 5, wherein the positions of said sleeve relative to said heat source for each desired temperature is determined empirically to provide an empirical formula, and wherein said temperature sensor in said sleeve which is operatively associated with an algorithm defining said empirical formula senses that a desired temperature is reached, instruct said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired temperature in the reaction vessel.

15. The thermal cycling system of claim 14, wherein, when said temperature sensor senses that the temperature in said reaction vessel approaches the desired template denaturation temperature, the algorithm defining said empirical formula instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired template denaturation temperature.

16. The thermal cycling system of claim 14, wherein, when said temperature sensor senses that the temperature in said reaction vessel approaches the desired primer annealing temperature, the algorithm defining said empirical formula instructs said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired primer annealing temperature.

17. The thermal cycling system of claim 14, wherein, when said temperature sensor senses that the temperature in said reaction vessel approaches the desired primer annealing temperature, the algorithm defining said empirical formula instruct said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired primer annealing temperature.

18. The thermal cycling system of claim 1, wherein said sleeve is provided with small openings that allow the samples inside said reaction vessel to be excited and imaged as part of a fluorescence detection apparatus.

19. The thermal cycling system of claim 1, wherein said reaction vessel includes a plug-style cap which is situated within said reaction vessel and wherein said sleeve extends up the sides of said reaction vessel, so that said plug will be heated and will minimize evaporation into the top of the vessel.

35. The method of claim 1, which comprises providing said sleeve with small openings that allow the samples inside the reaction vessel to be excited and imaged as part of a fluorescence detection apparatus.

36. The method of claim 1, which comprises minimizing evaporation into the top of said vessel by placing a plug-style cap reaction vessel into said reaction vessel and by positioning said sleeve to extend up the sides of the reaction vessel, so that said plug will be heated.

2. A thermal cycling system for performing a polymerase chain reaction amplification protocol comprising multiple cycles of three temperature-dependent stages of template denaturation, about 90°

C., primer annealing about 60°

C. and primer extension, about 68°

C. that constitute a single cycle of PCR, the thermal cycycling system comprising a) a heat source that is set at a fixed temperature;

b) a reaction vessel containing material upon which a polymerase chain reaction amplification protocol is to be performed;

c) mechanically-operable means for altering the relative position of the heat source and the reaction vessel so that, the temperature of the reaction vessel is achieved and is maintained for carrying out template denaturation on said material, and then for altering the relative position of the heat source and the reaction vessel so that, the temperature of the reaction vessel is achieved and is maintained for carrying out primer annealing on the material and then for altering the relative position of the heat source and the reaction vessel so that, the temperature of the reaction vessel is achieved and is maintained for carrying out primer extension on the material; and

d) temperature-sensing means operatively associated with the reaction vessel for controlling the altering of the relative position of the heat source and the reaction vessel so that the reaction vessel achieves and maintains the desired second temperature in the reaction vessel.

37. The thermal cycling system, of claim 2, wherein said heat source is a block of heat retentive material including means to heat said block to, and maintain said block at a fixed temperature.

38. The thermal cycling system of claim 37, wherein said reaction vessel is embedded in a metal sleeve, and wherein said metal sleeve is configured and arranged to be movable.

39. The thermal cycling system of claim 38, wherein said temperature sensor in said sleeve is operatively associated with a processor which is downloaded with an algorithm to predict the temperature being experienced by said reaction vessel, said algorithm being based on a program to achieve and maintain a desired temperature in the reaction vessel.

40. The thermal cycling system of claim 38, wherein the positions of said sleeve relative to said heat source for each desired temperature is determined empirically to provide an empirical formula, and wherein said temperature sensor in said sleeve which is operatively associated with an algorithm defining said empirical formula senses that a desired temperature is reached, instruct said moving means to change the relative position of said sleeve with respect to said block to attain and maintain said desired temperature in the reaction vessel.

20. A thermal cycler comprising at least one fixed-temperature heat source where additional lower temperatures may be achieved and maintained by positioning a reaction vessel or sleeve in close proximity to the heat source, but not in contact.

23. The method of claim 20, which comprises maintaining said heat source fixed in place and moving said reaction vessel.

24. The method of claim 20, which comprises moving said heat source and maintaining said reaction vessel fixed in place.

21. A method for performing a biological reaction at two or more different temperatures, the method comprising the steps of:

a) placing a reaction vessel containing a biological mixture in a position with respect to a heat source that is set at a fixed temperature to allow said reaction vessel to achieve and maintain a desired first temperature for starting the carrying out of said biological reaction;

b) relatively moving said reaction vessel with respect to said heat source, thereby to achieve and maintain a second temperate for continuing the carrying out of said biological reaction on the biological material; and

c) controlling the relative movement of said heat source and said reaction vessel by a temperature sensor which is operatively associated with said reaction vessel to achieve and maintain a desired reaction temperatures in said reaction vessel.

25. The method of claim 21, which further comprises the steps of embedding said reaction vessel in a metal sleeve, and providing said metal sleeve with a temperature sensor.

26. The method of claim 25, including the step of changing the relative position of said sleeve with respect to said block to attain and maintain said reaction vessel at a first desired template denaturation temperature when said temperature sensor senses that the temperature of said sleeve approaches said template denaturization temperature.

27. The method of claim 25, including the step of changing the relative position of said sleeve with respect to said block to attain and maintain the reaction vessel at a primer annealing temperature when said temperature sensor senses that the temperature of said sleeve approaches said desired reaction primer annealing temperature.

28. The method of claim 25, including the step of changing the relative position of the sleeve with respect to the block to attain and maintain the reaction vessel at a template denaturation temperature when said temperature sensor senses that the temperature of said sleeve approaches the desired template denaturation temperature.

29. The method of claim 26, which comprises the steps of providing a processor with an algorithm to predict the temperature being experienced by said reaction vessel, and changing the relative position of said sleeve with respect to said block to attain and maintain the temperature of said reaction vessel at a primer annealing temperature when said algorithm predicts that the temperature of said reaction vessel approaches a primer annealing temperature.

30. The method of claim 26, which comprises the steps of providing a processor with an algorithm to predict the temperature being experienced by said reaction vessel, and changing the relative position of said sleeve with respect to said block to attain and maintain temperature of said reaction vessel at a primer extension temperature when said algorithm predicts that the temperature of said reaction vessel approaches a primer extension temperature.

31. The method of claim 26, which comprises the steps of empirically determining the positions of said sleeve relative to said heat source for each desired temperature, providing an empirical formula thereof and converting said empirical formula into an algorithm, and changing the relative position of said sleeve with respect to said block to attain and maintain a desired temperature in said reaction vessel when said algorithm determines that the temperature of said reaction vessel approaches the desired temperature.

32. The method of claim 31, which comprises the steps of empirically determining the positions of said sleeve relative to said heat source for a desired template denaturation temperature, providing an empirical formula thereof and converting said empirical formula into an algorithm and changing the relative position of said sleeve with respect to said block to attain and maintain the desired template denaturation temperature in said reaction vessel when said algorithm determines that the temperature of said reaction vessel approaches the desired template denaturation temperature.

33. The method of claim 31, which comprises the steps of empirically determining the positions of said sleeve relative to said heat source for a desired primer annealing temperature, providing an empirical formula thereof and converting said empirical formula into an algorithm, and changing the relative position of said sleeve with respect to said block to attain and maintain a desired primer annealing temperature in said reaction vessel when said algorithm determines that the temperature of said reaction vessel approaches a desired primer annealing temperature.

34. The method of claim 31, which comprises the steps of empirically determining the positions of said sleeve relative to said heat source for a desired primer extension temperature, providing an empirical formula thereof and converting said empirical formula into an algorithm, and changing the relative position of said sleeve with respect to said block to attain and maintain a desired primer extension temperature in said reaction vessel when said algorithm determines that the temperature of said reaction vessel approaches a desired primer extension temperature.

22. A method for performing a polymerase chain reaction amplification protocol comprising multiple cycles of three sequential temperature-dependent stages that constitute a single cycle of PCR:

comprising template denaturation, primer annealing; and

primer extension on a biological material, the method comprising the steps of;

a) placing a reaction vessel containing said biological material in a position with respect to a heat source that is set at a fixed temperature to allow the reaction vessel to achieve and maintain a desired temperature for carrying out template denaturation;

b) relatively moving said reaction vessel with respect to said heat source, thereby to achieve a suitable temperature of said reaction vessel for carrying out primer annealing;

c) relatively moving said reaction vessel with respect to said heat source thereby to achieve a suitable temperature of said reaction vessel for carrying out primer extension; and

d) controlling the relative movement of said heat source and said reaction vessel by a temperature-sensor which is operatively associated with said reaction vessel to achieve and maintain the desired template denaturation, primer annealing; and

primer extension temperatures that constitute a single cycle of PCR in the reaction vessel