Thermal cycler including a temperature gradient block

• US 5,779,981 A
• Filed: 04/19/1996
• Issued: 07/14/1998
• Est. Priority Date: 10/20/1993
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Shopes, Robert James, Danssaert, John Lewis, Shoemaker, Daniel Davis
• Primary Examiner(s): Walsh, Stephen
• Assistant Examiner(s): LAZAR WESLEY, ELIANE M
• Application Number:
  US08/634,826
• Time in Patent Office: 816 Days
• Field of Search: 422/99, 422/67, 422/63, 422/104, 435/809, 435/285.1, 435/286.1, 435/286.2, 435/288.4, 435/303.1, 935/85
• US Class Current: 422/67
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1838 using fluid heat transfer medium
• CPC Class Codes: B01L 2300/1844 using fans
• 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 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: B01L 7/5255 by moving sample containers
• CPC Class Codes: B01L 7/54 using spatial temperature gradients
• CPC Class Codes: G01N 2035/042 moved independently, e.g. by fork manipulator
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: Y10S 435/809 Incubators or racks or holders for culture plates or containers
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

A method in which a temperature gradient is generated across a "gradient" block, and an apparatus comprising a block across which a temperature gradient can be generated. By setting up such a gradient, multiple reaction mixtures held in wells on the gradient block can be simultaneously run at temperatures which differ only slightly, thereby permitting an optimum temperature for the reaction to be quickly identified. In a preferred embodiment the gradient block is integrated into a thermal cycler used for nucleic acid amplification reactions.

Claims

1. A method for simultaneously reacting a plurality of reaction mixtures in an apparatus including a temperature gradient block and further including first and second uniform temperature blocks, said method comprising the steps of:

placing reaction mixtures in a plurality of reaction wells in said gradient block, said gradient block having first and second opposing end portions, said plurality of reaction mixture wells being formed in said block between said opposing end portions;

generating a temperature gradient across said gradient block and between said opposing end portions; and

placing the reaction mixtures in a plurality of reaction wells in said first uniform temperature block; and

placing the reaction mixtures in a plurality of reaction wells in said second uniform temperature block; and

generating uniform temperatures in said uniform temperature blocks.

2. A method according to claim 1 wherein the step of generating a temperature gradient is performed before the reaction mixtures are placed in the gradient block.

3. A method according to claim 1 wherein the step of placing reaction mixtures in the gradient block is performed before the step of placing reaction mixtures in the first uniform temperature block.

4. A method according to claim 1 wherein the step of placing reaction mixtures in the gradient block is performed after the step of placing reaction mixtures in the first uniform temperature block and before the step of placing reaction mixtures in the second uniform temperature block.

5. An apparatus for generating a temperature gradient across a heat conducting block comprising:

a reaction mixture holder including a heat conducting block having top and bottom portions, first and second opposing end portions, and a plurality of reaction mixture wells formed in said top portion;

a first block heater positioned adjacent to said first opposing end portion;

a block cooler positioned adjacent to said second opposing end portion;

a second block heater positioned adjacent to said bottom portion; and

means for independently controlling the first and second block heaters and the block cooler.

6. An apparatus according to claim 5 wherein said heat conducting block is composed of brass.

7. An apparatus according to claim 5 wherein said plurality of wells in said heat conducting block are spaced across said top portion in parallel, aligned rows.

8. An apparatus according to claim 5 wherein the block cooler is a heat sink attached to the second opposing end portion of the heat conducting block.

9. An apparatus according to claim 8 wherein the block cooler further comprises a fan positioned adjacent to said heat sink.

10. An apparatus for performing molecular biological reactions comprising:

a temperature-gradient block, said temperature-gradient block having a plurality of reaction mixture wells spaced between first and second opposing end portions thereof;

means for generating a temperature gradient between said first and second opposing end portions of the temperature-gradient block;

first and second uniform-temperature blocks, each having a plurality of reaction mixture wells; and

means for independently heating each uniform-temperature block.

11. An apparatus according to claim 10 wherein said temperature-gradient block is composed of brass.

12. An apparatus according to claim 10 further comprising a heating means for heating a bottom portion of said temperature-gradient block.

13. An apparatus according to claim 10 wherein said apparatus further comprises robot arm means, controlled by robot arm control means, for moving reaction mixture between the wells of the temperature-gradient block and the wells of the uniform-temperature block.

14. An apparatus according to claim 10 further comprising a third uniform-temperature block having a plurality of reaction mixture wells and means for cooling the third uniform-temperature block.

15. A method for determining an optimum temperature for one step of a multistep biological reaction process comprising the steps of:

generating a temperature gradient across a gradient-temperature block having a plurality of reaction wells, wherein the reaction wells are at different temperatures;

placing reaction mixtures in the reaction wells in the gradient-temperature block to subject the reaction mixtures to different temperatures as one step of the multistep biological reaction process;

generating a uniform temperature in a uniform-temperature block having a plurality of reaction wells;

placing the reaction mixtures in the reaction wells in the uniform-temperature block as another step of the multistep biological reaction process; and

analyzing the results of the multistep biological reaction process according to the different temperatures applied to the reaction mixtures in the gradient-temperature block to determine an optimum temperature for said one step.

16. A method according to claim 15 wherein the multistep biological reaction process is a polymerase chain reaction.

17. A method according to claim 16 wherein a denaturing step of a polymerase chain reaction is performed in the gradient-temperature block, and other steps of the polymerase chain reaction are performed in uniform-temperature blocks.

18. A method according to claim 16 wherein an annealing step of a polymerase chain reaction is performed in the gradient-temperature block, and other steps of the polymerase chain reaction are performed in uniform-temperature blocks.

19. A method according to claim 16 wherein an extension reaction step of a polymerase chain reaction is performed in the gradient-temperature block, and other steps of the polymerase chain reaction are performed in uniform-temperature blocks.

20. A method according to claim 17 wherein the multistep biological reaction process is a ligase chain reaction.

21. A method according to claim 20 wherein a high temperature melting step of the ligase chain reaction is performed in the gradient-temperature block, and a cooling step of the ligase chain reaction is performed in the uniform-temperature block.

22. A method according to claim 20 wherein a cooling step of the ligase chain reaction is performed in the gradient-temperature block, and a high temperature melting step of the ligase chain reaction is performed in the uniform-temperature block.