Nucleic acid amplification reaction apparatus and method

• US 6,033,880 A
• Filed: 02/23/1998
• Issued: 03/07/2000
• 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: Tracy, David, Haff, Lawrence A., Bloch, Will, Ragusa, Robert, Noreiks, Richard W., Picozza, Enrico, DiCesare, Joseph, Saviano, Paul, Woudenberg, Timothy M.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/027,989
• Time in Patent Office: 743 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 422/50, 422/68.1, 536/25.3, 935/77, 935/78, 436/501
• US Class Current: 435/91.1
• 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. A method for performing a nucleic acid amplification reaction including denaturation, annealing, and extension processes, simultaneously in a plurality of reaction mixtures contained in a plurality of capillary tubes, each mixture containing a target nucleic acid to be amplified, comprising the steps of:

(a) drawing the mixtures into first portions of the plurality of capillary tubes;

(b) heating the first portions to an extension temperature operative to cause the extension process to occur in the reaction mixtures;

(c) heating the first portion to a denaturation temperature in a range of temperatures operative to cause the denaturation process to occur in the reaction mixtures;

(d) cooling the first portions of the capillary tubes to a annealing temperature in a range of temperatures operative to cause the annealing process to occur in the reaction mixtures in the first portions of the capillary tubes; and

(e) repeating steps (b) through (d) a desired number of times to reach a desired quantity of the nucleic acids in the samples.

2. The method according to claim 1 wherein step (c) further comprises the steps of (i) subjecting the first portions of the capillary tubes to a temperature greater than the denaturation temperature only for a period necessary to cause the reaction mixtures to ramp to the denaturation temperature and then (ii) holding the denaturation temperature in the mixtures for a predetermined period of time for the denaturation process to occur in the mixtures.

3. The method according to claim 1 wherein the cooling step (d) comprises the steps of (i) subjecting the first portions of the capillary tubes to a temperature lower than the annealing temperature for a period of time effective to ramp the temperature of the reaction mixtures to the annealing temperature, and (ii) maintaining the first portions at the annealing temperature for a period of time effective to cause the annealing process to occur in the mixtures.

4. The method according to claim 2 wherein the cooling step (d) comprises the steps of (i) subjecting the first portions of the capillary tubes to a temperature lower than the annealing temperature for a period of time effective to ramp the temperature of the reaction mixtures to the annealing temperature, and (ii) maintaining the first portions at the annealing temperature for a period of time effective to cause the annealing process to occur in the mixtures.

5. A method of amplifying, by a nucleic acid amplification reaction, at least one target nucleic acid sequence in each one of a plurality of reaction mixtures, the PCR including denaturation, annealing, and extension processes, the method comprising the steps of:

(a) drawing the mixtures into first portions of a plurality of capillary tubes;

(b) positioning the mixtures in second portions of the capillary tubes each against a first surface portion of a movable heat exchanger, the first surface portion being maintained at a temperature suitable for causing the extension process to occur in the mixtures;

(c) moving the heat exchanger to a second position wherein a second surface portion of the heat exchanger contacts the second portions of the capillary tubes, the second surface portion having a temperature greater than the denaturation temperature;

(d) moving the heat exchanger to a third position wherein a third surface portion of the heat exchanger contacts the second portions of the capillary tubes, the third surface portion having a temperature in a range of temperatures effective to cause the denaturation process to occur in the mixtures;

(e) moving the heat exchanger to a fourth position wherein a fourth surface portion of the heat exchanger contacts the second portions of the capillary tubes, the fourth surface portion having a temperature in a range of temperatures suitable for annealing reactions to occur in the mixtures;

(f) repeating steps (b) through (e) a sufficient number of times to amplify the quantity of the target nucleic acids a desired amount.

6. The method of claim 5 wherein the moving step (d) further comprises the step of holding the movable heat exchanger at the third position for a predetermined period of time.

7. The method of claim 5 wherein the moving step (e) further comprises the step of holding the heat exchanger at the fourth position for a predetermined period of time.

8. The method of claim 6 wherein the moving step (e) further comprises the step of holding the heat exchanger at the fourth position for a predetermined period of time.

9. The method of claim 7 wherein the first surface portion and the second surface portion are the same.

10. The method of claim 8 wherein the first surface and portion second the surface portion are the same.

11. A method of amplifying, by a nucleic acid amplification reaction, at least one target nucleic acid sequence in each one of a plurality of reaction mixtures, the amplification reaction including denaturation, annealing, and extension processes, the method comprising the steps of:

(a) drawing the mixtures into first portions of a plurality of capillary tubes;

(b) positioning the mixtures in second portions of the capillary tubes each against a first surface portion of a rotatable heat exchanger, the first surface portion being maintained at a temperature suitable for causing the extension process to occur in the mixtures;

(c) rotating the heat exchanger about a central axis to a second position wherein a second surface portions of the heat exchanger contacts the second portion of the capillary tubes, the second surface portion having a temperature above a range of temperatures effective to cause the denaturation process to occur in the mixtures;

(d) rotating the heat exchanger to a third position wherein a third surface portion of the heat exchanger contacts the second portions of the capillary tubes, the third surface portion having a temperature in a range of temperatures effective to cause the denaturation process to occur in the mixtures;

(e) rotating the heat exchanger to a fourth position wherein a fourth surface portion of the heat exchanger contacts the second portions of the capillary tubes, the fourth surface portion having a temperature in a range of temperatures suitable for the annealing process to occur in the mixtures;

(f) repeating steps (b) through (e) a sufficient number of times to amplify the quantity of the target nucleic acid sequence a desired amount.

12. The method of claim 11 wherein the rotating step (d) further comprises the step of holding the rotatable heat exchanger at the third position for a predetermined period of time.

13. The method of claim 11 wherein the rotating step (e) further comprises the step of holding the heat exchanger at the fourth position for a predetermined period of time.

14. The method of claim 12 wherein the rotating step (e) further comprises the step of holding the heat exchanger at the fourth position for a predetermined period of time.

15. The method of claim 13 wherein the first surface portion and the second surface portion are the same.

16. The method of claim 14 wherein the first surface portion and the second surface portion are the same.

17. A method of automatically performing a nucleic acid amplification reaction in a reaction mixture in a capillary tube containing a target nucleic acid segment to be amplified, the amplification reaction including denaturation, annealing and extension processes, the method comprising the steps of:

(a) automatically moving the reaction mixture from a container into an open capillary tube reaction chamber having a portion adapted to contain the reaction mixture;

(b) heating the mixture in the capillary tube reaction chamber to a first temperature in a range of temperatures suitable to cause the denaturation process to occur in the mixture;

(c) cooling the mixture to a second temperature suitable to cause the annealing and extension processes to occur in the mixture;

(d) repeating steps (b) and (c) a user-defined number of times;

(e) automatically discharging the reaction mixture out of the capillary tube reaction chamber into the container;

(f) automatically moving a different reaction mixture from another container into the reaction chamber portion; and

(g) automatically repeating steps (b) through (e);

wherein during steps (b), (c), and (d) said reaction mixture is contained and is stationary within said portion of said open capillary tube reaction chamber adapted to contain said reaction mixture.