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

• US 5,038,852 A
• Filed: 03/14/1990
• Issued: 08/13/1991
• Est. Priority Date: 02/25/1986
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Cetus Corporation (Bayer AG)
• Inventors: Johnson, Larry J., Widunas, Joseph T.
• Primary Examiner(s): Wayner, William E.
• Application Number:
  US07/494,174
• Time in Patent Office: 517 Days
• Field of Search: 165/12, 236/46 R, 364/557, 73/863.11, 422/116, 436/50
• US Class Current: 165/267
• 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 performing temperature cycling of a reaction mix 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 means for heating and cooling is an aluminum plate having fluid flow channels formed therein which are in fluid communication with pumps which circulate fluid stored in fluid reservoirs having heating and cooling elements therein to keep the fluid at a constant, user-definable temperature.

2. An apparatus for performing temperature cycling of a reaction mix 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.

3. An apparatus for performing temperature cycling of a reaction mix 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 in said computer means for receiving and storing data for a number of cycles data field from the user for each set of checkpoints entered by the user to define a temperature profile and for running each temperature profile the number of times identified in said number of cycles data field before checking said link field of the temperature profile for the identification of the temperature profile to be run next.

4. An apparatus for performing automated amplification of at least one specific nucleic acid sequence comprising:

a first means for holding a reaction mixture comprising said nucleic acid sequence(s) to be amplified, four different nucleotide triphosphates, a thermostable enzyme, and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands of each specific sequence, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, said holding being carried out at any selected temperature or plurality of temperatures; and

a second means for automatically performing a predetermined sequence of steps including causing said first means to heat its contents for a first period and to cool its contents for a second period; and

wherein said second means includes means for allowing the user to enter data which control certain process parameters that characterize predetermined steps in said sequence of steps; and

wherein said first means holds said liquid stored therein at either of two temperatures and wherein said second second means causes said first means to hold its contents at a first temperature for a user-defined interval followed by a chill-down period and a low-temperature incubation at said second temperature having a user-defined duration; and

wherein said first means is a reaction chamber thermally coupled to an aluminum plate having fluid flow channels formed therein which are in fluid communication with pumps which circulate fluid stored in fluid reservoirs having heating and cooling elements therein to keep the fluid at a constant, user-definable temperature.