Nucleic acid amplification reaction apparatus

• US 5,720,923 A
• Filed: 08/31/1994
• Issued: 02/24/1998
• Est. Priority Date: 07/28/1993
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Woudenberg, Timothy M., Ragusa, Robert, Tracy, David, Haff, Lawrence A., Bloch, Will, Picozza, Enrico, DiCesare, Joseph, Saviano, Paul
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/299,033
• Time in Patent Office: 1,273 Days
• Field of Search: 422/50, 422/62, 422/63, 422/67, 422/68.1, 422/69, 422/81, 422/82.05, 422/82.08, 422/82.09, 422/129, 422/131, 422/132, 422/134, 422/138, 422/149, 422/159 , 422/187-189, 422/196, 422/197, 422/198, 422/209, 422/236, 435/6, 435/91.1, 435/91.2, 435/91.5, 435/287-289, 435/291, 435/296, 435/316, 435/283.1, 435/285.1, 435/285.2, 435/287.1, 435/287.2, 435/287.3 , 435/289.1, 435/290.1, 436/501, 935/78, 935/88
• 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 containing a target nucleic acid segment including denaturation, annealing and extension processes, said apparatus comprising:

a capillary tube reaction chamber having a portion adapted to contain the reaction mixture at a predetermined position;

first temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber for subjecting the reaction mixture at said position to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber at said position for subjecting the mixture to a second temperature suitable to cause the annealing and extension process to occur in the mixture;

sample handling means connected to the capillary tube reaction chamber for automatically moving the reaction mixture into and out of the capillary tube reaction chamber and to and from said predetermined position; and

positioning means for automatically positioning the first and second heat exchange means sequentially in contact with the portion of the capillary tube reaction chamber,

wherein each of the heating means is a segment of a cylindrical heat exchange drum.

2. The apparatus according to claim 1 further comprising a third temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube for subjecting the portion to a temperature above the first temperature prior to contacting said portion with said first temperature controlled heating means for a time sufficient to drive the temperature of the mixture up to said first temperature.

3. The apparatus according to claim 1 wherein the cylindrical heat exchange drum has at least two axially extending radial segments, each separated from each other by a layer of thermal insulation.

4. The apparatus according to claim 2 wherein each of the segments has an arcuate outer surface and a groove in the arcuate outer surface each adapted to receive the portion of the capillary tube reaction chamber.

5. The apparatus according to claim 4 wherein the drum is rotatable about its central axis between positions in which each of the segments contacts the portion of the capillary tube reaction chamber.

6. The apparatus according to claim 5 further comprising a programmable computer connected to the heating means and the rotatable drum operable to control the temperatures of the heat exchange means and the position of the drum in accordance with user-defined parameters.

7. The apparatus according to claim 1 further comprising a plurality of open capillary tube reaction chambers each having a portion adapted to receive a reaction mixture.

8. The apparatus according to claim 7 wherein each of the segments has an arcuate outer surface and a plurality of parallel grooves in the arcuate outer surface each adapted to receive one of said portions of the capillary tube reaction chambers.

9. The apparatus according to claim 8 wherein the drum is rotatable about its central axis between positions in which each of the segments contacts said portion of each of the capillary tube reaction chambers.

10. The apparatus according to claim 9 further comprising a programmable computer connected to the heating means and the rotatable drum and wherein the computer controls the rotatory position of the drum in accordance with user defined parameters.

44. The apparatus according to claim 1, wherein temperature of the segments is thermostatically controlled.

49. The apparatus according to claim 1, wherein the segments are radial segments.

11. An apparatus for performing a reaction in a reaction mixture containing a target nucleic acid segment including denaturation, annealing and extension processes in a capillary tube, the apparatus comprising:

a capillary tube reaction chamber for containing the reaction mixture;

first temperature controlled movable heat exchange means adapted to be coupled to the capillary tube for subjecting a portion of the capillary tube reaction chamber to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to be coupled to the capillary tube for subjecting said portion of the capillary tube to a second temperature suitable to cause the annealing and extension processes to occur in the reaction mixture;

means for automatically moving the reaction mixture into said portion of the capillary tube reaction chamber and discharging the reaction mixture from the capillary tube reaction chamber; and

control means for sequentially coupling said first and second heat exchange means to the capillary tube in accordance with a reaction protocol to cause said reaction to take place in said reaction mixture.

12. The apparatus according to claim 11 wherein the capillary tube reaction chamber has one open end.

13. The apparatus according to claim 11 wherein the means for automatically moving is a computer-controlled peristaltic pump.

14. An apparatus for simultaneously performing a nucleic acid amplification reaction including denaturation, annealing and extension processes in reaction mixtures in a plurality of capillary tubes, the apparatus comprising:

a plurality of open ended capillary tubes each having a portion adapted to contain a reaction mixture suitable for causing the nucleic amplification reaction to create copies of a target nucleic acid segment;

first temperature controlled movable heat exchange means for simultaneously subjecting said portion of each of the capillary tubes to a first temperature suitable to cause the denaturation process in the nucleic acid amplification reaction to occur in the mixtures;

second temperature controlled movable heat exchange means for simultaneously subjecting said portion of each of the capillary tubes to a second temperature suitable to cause the annealing and extension processes in the nucleic acid amplification reaction to occur in the mixtures;

positioning means for causing said temperature controlled movable heat exchange means to subject the reaction mixtures to the first and second temperatures in accordance with a predetermined sequence of steps;

plurality of reaction tubes corresponding to said capillary tubes; and

handling means for operatively connecting an open end of each of the capillary tubes to a corresponding reaction tube containing the reaction mixture and for cleansing the capillary tubes.

15. The apparatus according to claim 14 wherein the handling means includes a reaction tube array translation and elevation system.

16. The apparatus according to claim 14 wherein the handling means includes a system for inserting and removing each of the capillary tubes from each of the reaction tubes.

17. The apparatus according to claim 14 wherein the positioning means comprises a programmable computer controlling the handling means and the first and second temperature controlled heat exchange means in accordance with a user defined set of parameters.

18. An apparatus for performing a nucleic acid amplification reaction on a plurality of reaction mixtures contained in a plurality of capillary tubes simultaneously, the reaction including denaturation, annealing and extension processes to generate a nucleic acid amplification reaction product, the apparatus comprising:

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 nucleic acid amplification reaction in the plurality of reaction mixtures;

a second heat exchanger including another 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 in the nucleic acid amplification reaction to occur in the mixtures;

a plurality of capillary tubes each routed so that each has a portion in thermal contact with at least one of the first and second heat exchangers;

sample handling means coupled to each of the capillary tubes for drawing the reaction mixture into the tube and positioning said mixtures in said portions, and for discharging finished reaction product from each of the tubes; and

user controllable means for sequentially moving the first and second heat exchangers into thermal contact with the portions of the capillary tubes containing the reaction mixtures so as to cause the nucleic acid amplification reaction to take place in the mixtures in the capillary tubes.

19. The apparatus of claim 18 further comprising a detector means optically coupled to the capillary tubes for detecting optical changes in the mixtures.

20. The apparatus according to claim 19 wherein the detector means includes a photodiode array.

21. The apparatus according to claim 19 wherein the detector means is coupled to a spectrophotometer.

22. The apparatus according to claim 19 wherein the detector means detects a fluorescence change occurring in the mixture during a portion of the nucleic acid amplification reaction.

23. The apparatus according to claim 22 wherein the detector means includes a photodiode device or CCD detector coupled to a computer.

24. The apparatus according to claim 18 wherein the first and second heat exchangers are each portions of an elongated rotatable drum.

25. The apparatus according to claim 24 wherein each of the heat exchangers is an arcuate segment of the cylindrical drum, each segment having an arcuate outer surface.

26. The apparatus according to claim 25 wherein the surface has a plurality of parallel grooves receiving the portions of the plurality of capillary tubes.

27. The apparatus according to claim 26 wherein the heat exchanger segments are separated by an insulating layer.

28. The apparatus according to claim 27 wherein the grooves are circumferential.

29. The apparatus according to claim 28 wherein the means for positioning is a computer-controlled stepper motor coupled to the rotatable cylindrical drum, the motor rotating the drum between a first angular position in which the first heat exchanger is in thermal contact with each of the portions of the capillary tubes and a second angular position in which the second heat exchanger is in thermal contact with each of the portions of the capillary tubes.

30. The apparatus according to claim 28 wherein the motor is further operable to rotate the cylindrical drum to a third angular position wherein a third heat exchanger segment having a temperature greater than that of the first and second heat exchanger segments contacts the portions of the tubes.

31. The apparatus according to claim 29 wherein the motor is further operable to rotate the cylindrical drum to a fourth angular position wherein a fourth heat exchanger segment having a temperature less than that of the first, second or third heat exchanger segments contacts the portions of the tubes.

32. The apparatus according to claim 19 wherein the means for drawing has at least one syringe pump connected to the capillary tubes.

33. The apparatus according to claim 32 wherein the means for drawing further comprises a lift mechanism coupled to an open end of each of the capillary tubes for inserting and withdrawing the one end of each tube into and out of at least one sample container.

34. The apparatus according to claim 33 wherein the means for drawing further comprises a computer controlled stepper motor mechanically connected to an elevating clamping bar holding the ends of the tubes in a spaced arrangement.

35. The apparatus according to claim 34 further comprising a translation mechanism coupled to a stage operable to index the sample containers to and from a position in registry with at least one of the ends of the capillary tubes.

36. The apparatus according to claim 35 wherein the stage contains a cleaning solution container and the translation mechanism is further operable to position the stage controllably in a clean and rinse position wherein the tube ends are in registry with the cleaning solution container.

37. The apparatus according to claim 36 further comprising a programmable computer operably coupled to the first and second heat exchangers, the lift mechanism, the translation mechanism, the means for positioning, and the thermoregulating systems to automatically handle the reaction mixture and perform the nucleic acid amplification reaction on the reaction mixture in accordance with user defined parameters to generate the reaction product.

38. The apparatus according to claim 18 wherein the first and second heat exchangers are each metal blocks.

39. The apparatus according to claim 38 wherein each of the heat exchangers is a generally rectangular block.

40. The apparatus according to claim 39 wherein each of the heat exchangers has a flat surface having a plurality of parallel grooves receiving the portions of the plurality of capillary tubes.

41. The apparatus according to claim 40 wherein the heat exchangers are separated by an insulating layer.

42. The apparatus according to claim 41 wherein the insulating layer is air.

43. The apparatus according to claim 42 wherein the means for positioning is a computer controlled syringe pump coupled to each of the tubes, the pump moving the reaction mixtures between the heat exchangers in accordance with a user-defined sequence of steps to perform the nucleic acid amplification reaction.

45. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture containing a target nucleic acid segment including denaturation, annealing and extension processes, said apparatus comprising:

a capillary tube reaction chamber having a portion adapted to contain the reaction mixture at a predetermined position;

first temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber for subjecting the reaction mixture at said position to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber at said position for subjecting the mixture to a second temperature suitable to cause the annealing and extension process to occur in the mixture;

sample handling means connected to the capillary tube reaction chamber for automatically moving the reaction mixture into and out of the capillary tube reaction chamber and to and from said predetermined position;

positioning means for automatically positioning the first and second heat exchange means sequentially in contact with the portion of the capillary tube reaction chamber; and

a third heating means adapted to contact the portion of the capillary tube for subjecting the portion to a temperature above the first temperature.

46. An apparatus for performing a nucleic acid amplification reaction on a plurality of reaction mixtures contained in a plurality of capillary tubes simultaneously, the reaction including denaturation, annealing and extension processes to generate a nucleic acid amplification reaction product, the apparatus comprising:

a first heat exchanger including a thermoregulating system for stabilizing the temperature of the first exchanger at a temperature in a range of temperatures suitable to cause the denaturation process to occur in the nucleic acid amplification reaction in the plurality of reaction mixtures;

a second heat exchanger including another 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 in the nucleic acid amplification reaction to occur in the mixtures;

a plurality of capillary tubes each routed so that each has a portion in thermal contact with at least one of the first and second heat exchangers;

sample handling means coupled to each of the capillary tubes for drawing the reaction mixture into the tube and positioning said mixtures in said portions, and for discharging finished reaction product from each of the tubes; and

user controllable means for sequentially causing relative motion between the first and second heat exchangers and the portions of the capillary tubes containing the reaction mixture so as to cause the nucleic acid amplification reaction to take place in the mixtures in the capillary tubes.

47. The apparatus according to claim 46 wherein the first and second heat exchangers are each stationary metal blocks.

48. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture containing a target nucleic acid segment including denaturation, annealing and extension processes, said apparatus comprising:

a capillary tube reaction chamber having a portion adapted to contain the reaction mixture at a predetermined position;

first temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber for subjecting the reaction mixture at said position to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber at said position for subjecting the mixture to a second temperature suitable to cause the annealing and extension process to occur in the mixture;

sample handling means connected to the capillary tube reaction chamber for automatically moving the reaction mixture into and out of the capillary tube reaction chamber and to and from said predetermined position; and

positioning means for automatically positioning the first and second heat exchange means sequentially in contact with the portion of the capillary tube reaction chamber; and

a plurality of open capillary tube reaction chambers each having a portion adapted to receive a reaction mixture;

wherein each of the heating means is a thermostatically controlled segment of a cylindrical heat exchange drum.