System for automated performance of the polymerase chain reaction

• US 5,656,493 A
• Filed: 02/18/1994
• Issued: 08/12/1997
• Est. Priority Date: 03/28/1985
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Johnson, Larry, Widunas, Joseph T., Mullis, Kary B., Mezei, Louis M., Wennberg, Timothy J., Leath, Richard A.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/199,505
• Time in Patent Office: 1,271 Days
• Field of Search: 435/6, 435/183, 435/91.1, 435/91.2, 435/286.11, 435/287.1, 435/287.2, 435/285.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 935/76, 935/77, 935/85, 935/87, 935/88, 422/50, 422/62, 236/91 R, 236/97, 236/1 C, 236/15 BG
• US Class Current: 435/286.1
• 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: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

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. A thermal cycling system for performing a polymerase chain reaction amplification protocol comprising multiple cycles of the steps of thermal denaturation of double-stranded DNA, primer hybridization to single-stranded DNA, and template-dependent primer extension by a DNA polymerase, comprising:

at least one reaction mixture comprising at least one single- or double-stranded nucleic acid sequence to be amplified, four different deoxyribonucleotides, and a pair of oligodeoxyribonucleotide primers for each said at least one nucleic acid sequence to be amplified,

for each said at least one reaction mixture, a heat-conducting reaction chamber,

in thermal contact with each said at least one chamber, a variable temperature heating and cooling system, the temperature of said heating and cooling system being computer controllable, and

a user-initiable computer controllingly coupled to said heating and cooling system, said computer being programmed to vary the temperature of said heating and cooling system and thereby to vary the temperature of said at least one chamber in accordance with said polymerase chain reaction protocol upon initiation by a user.

2. The thermal cycling system of claim 1, wherein said multiple cycles of the polymerase chain reaction protocol include a repetitive cycle which includes denaturation in the range of from 90°

to 105°

C. for up to 4 minutes.

3. The thermal cycling system of claim 2, wherein said cycle is repeated at least 15 times.

4. The thermal cycling system of claim 1 comprising a plurality of reaction mixtures, each in a separate reaction chamber, and wherein said heating and cooling system includes a metal block having a plurality of recesses shaped to fit said chambers and fluid flow channels, a temperature-controlled cooling fluid reservoir, and controllable pumping apparatus for pumping a cooling fluid from said cooling fluid reservoir through said channels in said block.

5. The thermal cycling system of claim 4, wherein said multiple cycles of the polymerase chain reaction protocol include a repetitive cycle which includes bring said chamber to a hybridization temperature in the range of from 35°

to 65°

C. for from 0.5 to 5 minutes, followed by extension product synthesis at a temperature in the range of from 40°

to 80°

C. for from 0.5 to 40 minutes and then by heating said chamber to a denaturation temperature in the range of from 90°

to 105°

C. for from 0.5 to 4 minutes.

6. The thermal cycling system of claim 5, wherein said cycle is repeated at least 15 times.

7. The thermal cycling system of claim 4, wherein said multiple cycles of the polymerase chain reaction protocol include a repetitive cycle which includes bringing said chamber to a hybridization temperature in the range of from 35°

to 65°

C., followed by heating said chamber to a denaturation temperature in the range of from 90°

to 105°

C. for from 0.5 to 4 minutes.

8. The thermal cycling system of claim 7, wherein said cycle is repeated at least 15 times.

9. The thermal cycling system of claim 4, wherein said heating and cooling system comprises a temperature-controlled heating fluid reservoir and controllable pumping apparatus for pumping a heating fluid from said heating fluid reservoir through said channels in said block.

10. The thermal cycling system of claim 4, 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 said chamber in response to a transfer control signal.

11. The thermal cycling system of claim 4, wherein said heating and cooling system comprises in electrical heater.

12. The thermal cycling system of claim 1, wherein said heating and cooling system has the capability to cool said at least one reaction mixture to temperatures suitable for reactions utilizing E. coli DNA polymerase I.

13. The thermal cycling system of claim 1, wherein said heating and cooling system has the capability to cool said at least one reaction mixture to temperatures suitable for reactions utilizing Klenow fragment of E. coli DNA polymerase I.

14. The thermal cycling system of claim 1, wherein said steps of hybridization and extension are performed at the same temperature.

15. The thermal cycling system of claim 1, wherein said step of extension is performed at a temperature higher than said step of hybridization.

16. The thermal cycling system of claim 1 comprising a plurality of reaction mixtures and a plurality of reaction chambers, and wherein said heating and cooling system includes a metal block having a plurality of recesses shaped to fit said chambers and a Peltier device.

17. The thermal cycling system of claim 1, wherein said programmed computer is user-programmable in real time.