Apparatus and method for performing automated amplification of nucleic acid sequences and assays using heating and cooling steps

• US 5,333,675 A
• Filed: 02/22/1993
• Issued: 08/02/1994
• Est. Priority Date: 02/25/1986
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Johnson, Larry, Widunas, Joseph T., Mullis, Kary B., Mezei, Louis M., Wennberg, Timothy J., Leath, Richard A.
• Primary Examiner(s): Wayner, William E.
• Application Number:
  US08/021,624
• Time in Patent Office: 526 Days
• Field of Search: 236/46 R, 165/12, 165/27, 364/557, 422/116, 436/505.5, 73/116, 935/87
• US Class Current: 165/268
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: Y10T 436/115831 Condition or time responsive

Abstract

There is disclosed herein a machine for performing nucleic acid amplification under computer control. The machine utilizes any one of a number of heating and cooling systems under control of a host computer which directs the heating and cooling systems to heat and cool a reaction-chamber-containing heat exchanger at appropriate times in the process. The reaction chambers are pre-loaded with the nucleic acid(s) to be amplified, a thermostable enzyme to catalyze polymerization, specific oligonucleotide primers, and four different nucleotide triphosphates. Also disclosed is the process for the amplification chain reaction implemented by the machine, which utilizes a thermostable enzyme.

Claims

1. An apparatus for automatically performing the polymerase chain reaction, comprising:

a reaction chamber for containing a reaction mixture suitable for performing the polymerase chain reaction;

a heat exchanger medium in thermal contact with said reaction chamber;

a temperature alteration system in thermal contact with said heat exchanger medium;

a programmable thermal controller coupled to said temperature alteration system for receiving data defining the desired polymerase chain reaction protocol and for controlling said temperature alteration system so as to alter the temperature of said heat exchanger medium in accordance with said desired polymerase chain action protocol;

wherein said heat exchanger medium is a metal block having fluid flow channels therein and wherein said temperature alteration system includes at least two temperature controlled fluid baths holding fluids therein at at least two different temperatures suitable for performing the polymerase chain reaction denaturation, hybridization and extension reactions, and a pump and valve mechanism coupled to said programmable thermal controller such that fluid from said at least two baths could be alternately pumped through said metal block heat exchanger.

2. An apparatus for automated temperature cycling of a plurality of reaction wells comprising:

a heat conducting metal block having a top surface, having a plurality of recesses communicating with said top surface for said plurality of reaction wells and having fluid flow channels therethrough,

a temperature-controlled cooling fluid reservoir,

controllable pumping means for pumping cooling fluid from said cooling-fluid reservoir through said fluid flow channels,

controllable means for heating said metal block,

a control input for controlling said controllable pumping means and said controllable means for heating, and

a computer means, coupled to the control input, for receiving and storing checkpoint data from the user defining a plurality of temperatures and times at which the temperatures are to be attained, thereby defining a temperature profile, and for, upon receipt of a command from the user, accessing the checkpoint data and generating control signals therefrom at the control input to cause the user-defined temperature profile to be achieved.

3. The apparatus of claim 2 wherein said controllable means for heating comprises a temperature-controlled heating fluid reservoir and controllable pumping means for pumping heating fluid from said heating fluid reservoir through fluid flow channels in said block.

4. The apparatus of claim 3 wherein at least one of said user-defined temperatures is below ambient.

5. The apparatus of claim 2 wherein the heat conducting metal block comprises aluminum.

6. The apparatus of claim 2 wherein the controllable means for heating comprises electrical heating means.

7. An apparatus according to claim 2 further comprising a computer-controlled liquid handler having at least one reagent container and pipettes for transferring reagent from said at least one reagent container into at least one sample tube resting in at least one of said wells in response to a transfer control signal, wherein said computer means includes programmable means for generating said transfer control signal.

8. Apparatus according to claim 2 further comprising liquid handling means controlled by said computer means and a second metal block, wherein said liquid handling means includes means for transferring liquid from one block to the other.

9. Apparatus according to claim 8 further comprising means to control the temperature of said second block.

10. Apparatus according to claim 9 wherein said second metal block has second fluid flow channels therethrough and wherein the means to alter the temperature of said second block includes means to flow fluid through said second fluid flow channels.

11. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

a heat-conducting container for holding a reaction mixture;

means for heating and cooling said container to or at any of a plurality of temperatures and having a control input for receiving a control signal controlling whether said container is heated or cooled;

a computer means, coupled to said control input of said means for heating and cooling, comprising means for receiving, storing and accessing a plurality of checkpoints from a user, each said checkpoint comprising a first time, said first time being a function of the time for which said container is to be maintained at one temperature of said plurality of temperatures, said accessing a plurality of checkpoints comprising generating control signals therefrom at the control input of said means for heating and cooling to cause said temperature to be achieved at said container and maintained for said time for which said container is to be maintained at said one temperature;

wherein said computer means further comprises user-controllable means for arranging said checkpoints in a sequence in which they are to be automatically accessed upon a command from the user, and user-controllable means for defining at least one subset of sequenced checkpoints, where said subset is less than the total number of checkpoints which will be accessed in sequence, which can be repeated a user-defined number of times before the checkpoint following the last checkpoint in the subset of sequenced checkpoints is accessed.

12. Apparatus of claim 11, wherein said first time equals said time for which said container is to be maintained at said one temperature.

13. Apparatus of claim 11, wherein said temperatures are user-definable.

14. Apparatus according to claim 11 further comprising liquid handling means controlled by said computer means.

15. Apparatus according to claim 11 wherein said means for heating and cooling said container is capable of cooling said container to sub-ambient temperatures.

16. Apparatus according to claim 11 wherein said means for heating and cooling said container includes a metal block supporting said containers.

17. The apparatus of claim 11, wherein said means for heating and cooling said container comprises a Peltier device.

18. The apparatus of claim 11, wherein said means for cooling said container comprises a Peltier device.

19. The apparatus of claim 11, wherein said means for cooling said container comprises a thermoelectric cooler.

20. The apparatus of claim 11, wherein said means for heating said container comprises an electrical heater.

36. The apparatus of claim 11, 33 or 34, wherein said means for cooling comprises means for cooling to temperatures suitable for reactions utilizing E. coli DNA polymerase I.

37. The apparatus of claim 11, 33 or 34, wherein said means for cooling comprises means for cooling to temperatures suitable for reactions utilizing Klenow fragment of E. coli DNA polymerase I.

38. The apparatus of claim 11, 33 or 34 wherein said means for cooling said container comprises means for pumping a cooled fluid such that heat is transferred from the container to the fluid.

39. The apparatus of claim 11, 33 or 34, wherein said means for heating said container comprises means for pumping a heated fluid such that heat is transferred from the fluid to the container.

21. An apparatus for automated temperature cycling of a plurality of reaction wells comprising:

a heat conducting metal block having a top surface, having a plurality of recesses communicating with said top surface for said plurality of reaction wells and having fluid flow channels therethrough,

a temperature-controlled cooling fluid reservoir,

controllable pumping means for pumping cooling fluid from said cooling-fluid reservoir through said fluid flow channels,

controllable means for heating said metal block,

a control input for controlling said controllable pumping means and said controllable means for heating, and

a computer means, coupled to said control input, comprising means for receiving, storing and accessing a plurality of checkpoints from a user, each said checkpoint comprising a first time, said first time being a function of a second time, said second time being the time for which said container is to be maintained at one said temperature, and means for, upon accessing said checkpoints, generating control signals therefrom at the control input to cause said temperature to be achieved at said container and maintained for said second time,

wherein said computer means further comprises user-controllable means for arranging said checkpoints in a sequence in which they are to be automatically accessed upon a command from the user, and user-controllable means for defining at least one subset of sequenced checkpoints, where said subset is less than the total number of checkpoints which will be accessed in sequence, which can be repeated a user-defined number of times before the checkpoint following the last checkpoint in the subset of sequenced checkpoints is accessed.

22. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

means for heating and cooling a heat-conducting container for holding a reaction mixture to or at any of a plurality of temperatures and having a control input for receiving a control signal controlling whether said container is heated or cooled; and

a computer means, coupled to said control input of said means for heating and cooling, comprising means for receiving and storing a plurality of checkpoints from a user, thereby defining a temperature profile, each said checkpoint defining a first time for which said container is to be maintained at a first temperature, and means for, upon a command from the user, accessing said temperature profile thereby causing, for each said checkpoint in said temperature profile, a control signal to be generated at the control input of said means for heating and cooling to cause said first temperature to be achieved at said container and maintained for said first time;

wherein said computer means includes means to define a plurality of temperature profiles; and

further comprising user-controllable means to link a plurality of said temperature profiles in a sequence to form a protocol such that, upon a command from the user, the set of said temperature profiles in said protocol will be automatically accessed in said sequence; and

further comprising means to cycle a first said temperature profile a user-defined number of times, such that said first temperature profile will be accessed said user-defined number of times before completion or, if said first temperature profile is a non-terminal profile in a said protocol, before the next profile in the protocol is accessed.

23. The apparatus of claim 22, wherein said first temperature is user-definable.

24. The apparatus of claim 23, wherein said checkpoint data defines said first temperature.

25. The apparatus of claim 22, wherein said means for cooling said container comprises a Peltier device.

26. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

a heat-conducting container for holding a reaction mixture;

means for heating and cooling said container to or at any of a plurality of user-defined temperatures and having a control input for receiving a control signal controlling whether said container is heated or cooled; and

a computer means, coupled to said control input of said means, for receiving and storing checkpoint data from the user defining the plurality of temperatures and the times at which said temperatures are to be attained thereby defining a temperature profile, and for, upon receipt of a command from the user, accessing said checkpoint data and generating control signals therefrom at the control input of said means for heating and cooling to cause the user-defined temperature profile to be achieved at said container; and

wherein said computer means includes means for receiving and storing in a link data field in a database associated with each said temperature profile stored by said computer means link data entered by the user for every set of checkpoints defining a temperature profile, and for receiving and storing a plurality of sets of checkpoints input by the user to define a plurality of temperature profiles, each of which has its own link data item, and wherein said computer means also includes means to run any particular temperature profile identified, if any is identified, in the link data field of the temperature profile just run and to continue this process of running the temperature profiles identified in the link data fields associated with each temperature profile run until no more temperature profiles are identified; and

further comprising means for cycling a first said temperature profile a user-defined number of times, such that the first temperature profile will be achieved at said container said user defined number of times before checking said link field of the first temperature profile for the identification of the temperature profile to be run next.

27. The apparatus of claim 26, wherein said means for heating and cooling said container comprises a Peltier device.

28. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

means for heating and cooling a heat-conducting container for holding a reaction mixture to or at any of a plurality of user defined temperatures;

a computer means for receiving and storing checkpoint data from the user defining the plurality of temperatures and the times at which said temperatures are to be attained thereby defining a temperature profile, and means for, upon receipt of a command from the user, controlling said means for heating and cooling to cause the temperature profile to be achieved at said container; and

wherein said computer means includes means for receiving and storing a plurality of sets of checkpoints input by the user to define a plurality of temperature profiles, and wherein said computer means comprises means for executing said profiles, said executing comprising causing said profiles to be achieved at said container at least once, and wherein said computer means comprises means for receiving and storing profile linking data defining an execution sequence, said execution sequence comprising a sequenced set of said temperature profiles which will be executed in sequence automatically upon command from the user, and wherein each position in said execution sequence constitutes an execution sequence position; and

further comprising means for cycling at least one first said temperature profile in at least one execution sequence position n a user-defined number of times, such that said first temperature profile will be achieved at said container said user-defined number of times, when said first temperature profile is executed in said execution sequence position n.

29. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

a heating and cooling system for heating and cooling a heat-conducting container for holding a reaction mixture to or at any of a plurality of user defined temperatures;

a computer system for receiving and storing checkpoint data from the user defining the plurality of temperatures and the times at which said temperatures are to be attained thereby defining a temperature profile, and an interface between said computer system and said heating and cooling system for, upon receipt of a command from the user, controlling said heating and cooling system to cause the temperature profile to be achieved at said container; and

wherein said computer system comprises means for receiving and storing a plurality of sets of checkpoints input by the user to define a plurality of temperature profiles, and wherein said computer system comprises means for executing said profiles, said executing comprising causing said profiles to be achieved at said container at least once, and wherein said computer system comprises means for receiving and storing profile linking data defining an execution sequence, said execution sequence comprising a sequenced set of said temperature profiles which will be executed in sequence automatically upon command from the user; and

further comprising means for cycling a first said temperature profile a user-defined number of times, such that the first temperature profile will be achieved at said container said user-defined number of times, when said first temperature profile is executed.

30. The apparatus of claim 29, wherein said means for heating said container comprises an electrical heater.

31. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

means for heating and cooling a heat-conducting container for holding a reaction mixture to or at any of a plurality of user defined temperatures;

a computer means for receiving and storing checkpoint data from the user defining the plurality of temperatures and the times at which said temperatures are to be attained thereby defining a temperature profile, and means for, upon receipt of a command from the user, controlling said means for heating and cooling to cause the temperature profile to be achieved at said container; and

wherein said computer means includes means for receiving and storing a plurality of sets of checkpoints input by the user to define a plurality of temperature profiles, and wherein said computer means comprises means for running said profiles, said running comprising causing said profiles to be achieved at said container, and wherein said computer means also includes means for receiving and storing profile linking data defining one or more sequences in which temperature profiles will be run automatically upon the command from the user; and

further comprising means in said computer means for receiving and storing cycling data defining, for a first temperature profile, the number of times to automatically repeat a run when the command to run said first temperature profile is issued, and for running said first temperature profile the number of times defined by said cycling data prior to either running the next temperature profile in a said sequence, if said first temperature profile is a non-terminal profile in a said sequence, or termination.

32. The apparatus of claim 31, wherein the rate at which said container is heated or cooled is adjustable.

33. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

a heat-conducting container for holding a reaction mixture;

means for heating and cooling said container to or at any of a plurality of temperatures and having a control input for receiving a control signal controlling whether said container is heated or cooled;

a computer means, coupled to said control input of said means for heating and cooling, comprising means for receiving, storing and accessing a plurality of checkpoints from a user, each said checkpoint defining a first time for which said container is to be maintained at a first temperature, said accessing a plurality of checkpoints comprising generating control signals therefrom at the control input of said means for heating and cooling to cause said first temperature to be achieved at said container and maintained for said first time;

wherein said computer means further comprises means for arranging said checkpoints in a sequence in which they are to be automatically accessed, and means for defining at least one subset of sequenced checkpoints, where said subset is less than the total number of checkpoints which will be accessed in sequence, which can be repeated a number of times before the checkpoint subsequent to the last checkpoint in the subset of sequenced checkpoints is accessed.

34. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

a heat-conducting container for holding a reaction mixture;

means for heating and cooling said container to or at any of a plurality of temperatures and having a control input for receiving a control signal controlling whether said container is heated or cooled;

a computer means, coupled to said control input of said means for heating and cooling, comprising means for receiving, storing and accessing a plurality of checkpoints from a user, each said checkpoint defining a first temperature at which said container is to be maintained for a first time, said accessing a plurality of checkpoints comprising generating control signals at the control input of said means for heating and cooling to cause said first temperature to be achieved at said container and maintained for said first time;

wherein said computer means further comprises user-controllable means for arranging said checkpoints in a sequence in which they are to be automatically accessed, and user-controllable means for defining at least one subset of sequenced checkpoints, where said subset is less than the total number of checkpoints which will be accessed in sequence, which can be repeated a user-defined number of times before the checkpoint following the last checkpoint in the subset of sequenced checkpoints is accessed.

35. The apparatus of claim 34, wherein each said checkpoint defines said first time.

40. Apparatus capable of cycling a reaction mixture in a polymerase chain reaction process comprising:

a heat-conducting container for holding a reaction mixture;

means for heating and cooling said container to or at any of a plurality of temperatures and having a control input for receiving a control signal controlling whether said container is heated or cooled;

a computer means, coupled to said control input of said means for heating and cooling, comprising means for receiving and storing a plurality of data input from a user corresponding to a temperature cycling profile, said cycling profile including heating, cooling and temperature maintaining steps and for accessing said plurality of said stored data and generating control signals therefrom at the control input of said means for heating and cooling to cause said temperature cycling profile to be achieved at said container;

wherein said computer means further comprises user-controllable means for configuring said temperature cycling profile as a sequence of heating, cooling and temperature maintaining steps which are to be automatically accessed upon a command from the user, and user-controllable means for defining at least one subset of sequenced steps, where said subset is less than the total number of steps which will be accessed in sequence, which can be repeated a user-defined number of times before the step following the last step in the subset or sequenced steps if accessed.

41. Apparatus of claim 40 wherein the user-input data comprises at least one checkpoint defining a duration for each step.

42. Apparatus of claim 40, wherein said temperatures are user-definable.

43. Apparatus according to claim 40 wherein said means for heating and cooling said container is capable of cooling said container to sub-ambient temperatures.

44. Apparatus according to claim 40 wherein said means for heating and cooling said container includes a metal block supporting said container.

45. Apparatus according to claim 44 wherein said metal block further comprise a plurality of receptacles for supporting a plurality of containers.

46. The apparatus of claim 40, wherein said means for heating said container comprises an electrical heater.

47. The apparatus of claim 40 wherein said means for cooling said container comprises means for pumping a cooled fluid such that heat is transferred from the container to the fluid.

48. Apparatus of claim 40 wherein said computer means comprises a sequence of heating, cooling and temperature maintaining steps which will be automatically accessed upon a command from the user, said sequence including at least one subset of sequenced steps, where said subset is less than the total number of steps which will be accessed in sequence, which will be repeated a user-defined number of times before the step following the last step in the subset of sequenced steps if accessed.

49. Apparatus for performing temperature cycling of a material comprising:

a receptacle for supporting the material;

a source of thermal energy responsive to a first control signal for heating the material;

a sink of thermal energy responsive to a second control signal for cooling the material;

a memory means for receiving user input data defining a plurality of temperature profiles to be applied in a sequence for temperature cycling of the material, said user input data defining each temperature profile as a series of one or more steps selected, in any order, from among heating, cooling, and temperature maintaining steps, a link data item identifying the next temperature profile in the sequence of profiles to be applied, and, if one temperature profile is to be repeated a user defined number of cycles before the next temperature profile in the sequence is to be applied, a cycle data item identifying the number of times the one temperature profile is to be repeated; and

a computer means for accessing said memory means and providing said first and second control signals for controlling the source and sink of thermal energy to achieve said user defined temperature cycling at the receptacle.

50. The apparatus of claim 49 wherein the receptacle comprises a heat exchange body having a recess.

51. The apparatus of claim 50 wherein the recess in the heat exchange body is shaped to receive the material, said material being a tube.

52. The apparatus of claim 51 wherein the heat exchange body is a block of material containing fluid flow channels, the thermal sink comprises a cooling fluid and a pump for passing said cooling fluid through said block flow channels, and wherein the computer means controls the pump.

53. The apparatus of claim 52 wherein the thermal source comprises an electric heater.

54. The apparatus of claim 49 wherein the thermal sink comprises a refrigeration device.

55. The apparatus of claim 49 wherein the thermal source comprises an electric heater.

56. Apparatus of claim 49 wherein said memory means includes a defined plurality of temperature profiles to be applied in a sequence for temperature cycling of the material, each temperature profile being a series of one or more steps selected, in any order, from among heating, cooling, and temperature maintaining steps, a link data item identifying the next temperature profile in the sequence of profiles to be applied, and a cycle data item identifying the number of times the one temperature profile is to be repeated.

57. Apparatus according to claim 56 wherein said computer means includes means for receiving input from a user and in response thereto controlling the source and sink of thermal energy to achieve said defined sequence of temperature profiles at the receptacle.