Nucleic acid amplification reaction apparatus

• US 5,827,480 A
• Filed: 03/25/1997
• Issued: 10/27/1998
• Est. Priority Date: 07/28/1993
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: The Perkin-Elmer Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Woudenberg, Timothy M., Haff, Lawrence A., Bloch, Will, Picozza, Enrico
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/823,735
• Time in Patent Office: 581 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 435/283.1, 435/286.1, 435/286.2, 435/286.5, 435/287.1, 435/287.2, 435/287.3, 435/289.1, 435/293.1, 422/50, 422/68.1, 422/62, 422/63, 422/67, 422/69, 422/82.05, 935/77, 935/78
• US Class Current: 422/68.1
• 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 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/1883 using thermal insulation
• 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 9/065 specially adapted for capillary tubes
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: Y10T 436/116664 with automated titrator

Abstract

Apparatus and method for performing a nucleic acid amplification reaction and preferably a polymerase chain reaction (PCR) in a reaction mixture in at least one capillary tube. Several different embodiments are disclosed. One embodiment cycles a sample through a capillary tube loop passing through two thermostatted fluid baths. Another embodiment has the capillary tube routed alternatingly between two heat exchangers to that the sample makes only one pass through the tube. Other embodiments maintain the heat exchangers stationary and translate the samples between them. Still further embodiments maintain the samples stationary and either automatically translate or rotate the heat exchangers past the samples contained within the capillary tubes to perform the thermal cycles necessary for the amplification reaction.

Claims

1. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture in a capillary tube, the nucleic acid amplification reaction including denaturation, annealing and extension processes, the apparatus comprising:

(a) a first heat exchanger including a thermoregulating system for stabilizing the temperature of the first heat exchanger at a temperature in a range of temperatures suitable to cause the denaturation process to occur in the reaction mixture;

(b) a second heat exchanger including a thermoregulating system for stabilizing the temperature of the second heat exchanger at a temperature in a range of temperatures suitable for causing the annealing and extension processes to occur in the reaction mixture;

(c) the capillary tube being routed so as to have a first portion in thermal contact with the first heat exchanger and a second portion in thermal contact with the second heat exchanger;

(d) a valve assembly having an input port coupled to the capillary tube for receiving said reaction mixture into the tube and an output port for delivering a finished reaction product from the tube, the valve assembly allowing injection of new reaction mixture in a first position, allowing the reaction mixture to circulate in the capillary tube between the first and second heat exchangers in a second position, and allowing ejection of finished reaction product in a third position; and

(e) a pumping device fixedly disposed in one of the heat exchangers coupled to the capillary tube so as to circulate the reaction mixture in the capillary tube such that the reaction mixture passes sequentially and cyclically through the first and second heat exchangers.

2. The apparatus of claim 1 further comprising a spectrophotometric detector optically coupled to the capillary tube for generating data representing changes in the reaction mixture during the nucleic acid amplification reaction.

3. The apparatus of claim 1 further comprising a valve means coupled to the capillary tube for providing an entry port through which reagents, additional primer, or additional enzyme may be added to the reaction mixture in the capillary tube.

4. The apparatus of claim 1 further comprising a means for injecting a discontinuity adjacent to the reaction mixture in the capillary tube.

5. The apparatus of claim 4 further comprising a cycle counter coupled to the capillary tube for counting the passage of the discontinuity.

6. The apparatus of claim 1 further comprising a computer-directed controller coupled to the pumping device and to the valve assembly for generating control signals to the pumping device and the valve assembly to automatically implement a nucleic acid amplification reaction protocol in accordance with data stored in the computer-directed controller.

7. The apparatus of claim 6 wherein the computer directed controller is user-programmable such that data defining different nucleic acid amplification reaction protocols may be entered by a user.

8. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture in a capillary tube, the nucleic acid amplification reaction including denaturation, annealing and extension processes, the apparatus comprising:

(a) a first heat exchanger including a thermoregulating system for stabilizing the temperature of the first heat exchanger at a temperature in a range of temperatures suitable to cause the denaturation process to occur in the reaction mixture;

(b) a second heat exchanger including a thermoregulating system for stabilizing the temperature of the second heat exchanger at a temperature in a range of temperatures suitable for causing the annealing and extension processes to occur in the reaction mixture;

(c) the capillary tube being routed so as to have a first portion in thermal contact with the first heat exchanger and a second portion in thermal contact with the second heat exchanger;

(d) a valve assembly having an input port coupled to the capillary tube for receiving said reaction mixture into the tube and an output port for delivering a finished reaction product from the tube, the valve assembly allowing injection of new reaction mixture in a first position, allowing the reaction mixture to circulate in the capillary tube between the first and second heat exchangers in a second position, and allowing ejection of finished reaction product in a third position;

(e) a pumping device in one of the heat exchangers coupled to the capillary tube so as to circulate the reaction mixture in the capillary tube such that the reaction mixture passes sequentially and cyclically through the first and second heat exchangers; and

(f) a spectrophotometric detector optically coupled to the capillary tube for generating data representing changes in the reaction mixture during nucleic acid amplification reaction.

9. The apparatus of claim 8 further comprising a valve means coupled to the capillary tube for providing an entry port through which reagents, additional primer, or additional enzyme may be added to the reaction mixture in the capillary tube.

10. The apparatus of claim 8 further comprising a means for injecting a discontinuity adjacent to the reaction mixture in the capillary tube.

11. The apparatus of claim 10 further comprising a cycle counter coupled to the capillary tube for counting the passage of the discontinuity.

12. The apparatus of claim 8 further comprising a computer-directed controller coupled to the pumping device and to the valve assembly for generating control signals to the pumping device and the valve assembly to automatically implement a nucleic acid amplification reaction protocol in accordance with data stored in the computer-directed controller.

13. The apparatus of claim 12 wherein the computer directed controller is user-programmable such that data defining different nucleic acid amplification reaction protocols may be entered by a user.

14. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture in a capillary tube, the nucleic acid amplification reaction including denaturation, annealing and extension processes, the apparatus comprising:

(a) a first heat exchanger including a thermoregulating system for stabilizing the temperature of the first heat exchanger at a temperature in a range of temperatures suitable to cause the denaturation process to occur in the reaction mixture;

(b) a second heat exchanger including a thermoregulating system for stabilizing the temperature of the second heat exchanger at a temperature in a range of temperatures suitable for causing the annealing and extension processes to occur in the reaction mixture;

(c) the capillary tube being routed so as to have a first portion in thermal contact with the first heat exchanger and a second portion in thermal contact with the second heat exchanger;

(d) a valve assembly having an input port coupled to the capillary tube for receiving said reaction mixture into the tube and an output port for delivering a finished reaction product from the tube, the valve assembly allowing injection of new reaction mixture in a first position, allowing the reaction mixture to circulate in the capillary tube between the first and second heat exchangers in a second position, and allowing ejection of finished reaction product in a third position;

(e) a pumping device in one of the heat exchangers coupled to the capillary tube so as to circulate the reaction mixture in the capillary tube such that the reaction mixture passes sequentially and cyclically through the first and second heat exchangers;

(f) means for injecting a discontinuity adjacent to the reaction mixture in the capillary tube; and

(g) a cycle counter coupled to said capillary tube for counting the passage of said discontinuity.

15. The apparatus of claim 14 further comprising a valve means coupled to the capillary tube for providing an entry port through which reagents, additional primer, or additional enzyme may be added to the reaction mixture in the capillary tube.

16. The apparatus of claim 14 further comprising a computer-directed controller coupled to the pumping device and to the valve assembly for generating control signals to the pumping device and the valve assembly to automatically implement a nucleic acid amplification reaction protocol in accordance with data stored in the computer-directed controller.

17. The apparatus of claim 16 wherein the computer directed controller is user-programmable such that data defining different nucleic acid amplification reaction protocols may be entered by a user.

18. The apparatus of claim 14 wherein said cycle counter is fixedly disposed in one of said heat exchanges.

19. The apparatus of claim 14 wherein said cycle counter is a photo-electric sensor.

20. The apparatus of claim 14 wherein said cycle counter is a capacitance sensor.