METHOD FOR RAPID THERMAL CYCLING OF BIOLOGICAL SAMPLES

• US 20010007759A1
• Filed: 08/11/1998
• Published: 07/12/2001
• Est. Priority Date: 06/04/1990
• Status: Active Grant

• Current Assignee: Carl T. Wittwer, David R. Hillyard
• Original Assignee: Carl T. Wittwer, David R. Hillyard
• Inventors: HILLYARD, DAVID R., WITTWER, CARL T.
• Granted Patent: US 6,787,338 B2
• Time in Patent Office: Days
• US Class Current: 435/91.1
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2300/1872 Infrared light
• 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 9/065 specially adapted for capillary tubes
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 30/02 Column chromatography
• CPC Class Codes: G01N 30/06 Preparation

Abstract

A thermal cycling method and device is disclosed. The device comprises a sample chamber whose temperature can be rapidly and accurately modulated over a range of temperatures needed to carry out a number of biological procedures, such a the DNA polymerase chain reaction. Biological samples are placed in glass micro capillary tubes and then located inside the sample chamber. A programmable controller regulates the temperature of the sample inside the sample chamber. Once a heating cycle is completed, the controller opens a door to the chamber for venting hot air out and cool ambient air is moved in. Temperature versus time profiles corresponding to optimum denaturation, annealing and elongation temperatures for amplification of DNA are achieved by the present invention.

Claims

1. A method of subjecting at least a first biological sample to rapid thermal cycling, the method comprising the steps of:

(a) placing the first biological sample in at least a first container;

(b) raising the temperature of the first biological sample from a first temperature to a second temperature at a first rate at least as great as 1°

C. per second, the first temperature being at least 20°

C. different than the second temperature;

(c) holding the first biological sample at a temperature at least as great as the second temperature for not more than a first holding period, the first holding period being not greater than ten seconds;

(d) lowering the temperature of the first biological sample from the second temperature to at least the first temperature at a second rate at least as great as 1°

C. per second;

(e) holding the first biological sample at a temperature at least as low as the first temperature for not more than a second holding period, the second holding period being not greater than ten seconds; and

(f) removing the first biological sample from the first container.

2. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the first temperature is at least 30°

C. different than the second temperature.

3. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the first temperature is at least 40°

C. different than the second temperature.

4. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the first rate is at least as great as 4°

C. per second.

5. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 4

wherein the second rate is at least as great as 4°

C. per second.

6. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the first rate is at least as great as 10°

C. per second.

7. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 6

wherein the second rate is at least as great as 10°

C. per second.

8. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the first holding period is not greater than 3 seconds.

9. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 8

wherein the second holding period is not greater than 3 seconds.

10. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the first holding period is not greater than 1 second.

11. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 10

wherein the second holding period is not greater than 1 second.

12. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the step of placing the first biological sample in at least a first container comprises the steps of:

placing a first portion of the biological sample into the first container;

placing a second portion of the biological sample into a second container;

placing a third portion of the biological sample into a third container; and

placing a fourth portion of the biological sample into a fourth container.

13. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 12

further comprising the step of positioning the first, second, third, and fourth containers in a sample chamber and wherein the step of raising the temperature of the first biological sample comprises the step of subjecting the first, second, third, and fourth containers to infrared radiation.

14. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

further comprising the step of repeating steps (b) through (e) at least fifteen times.

15. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

further comprising the step of repeating steps (b) through (e) at least thirty times in not more than 10 minutes.

16. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the step of placing the first biological sample in at least a first container comprises the step of placing the first biological sample in at least the first container having a volume in the range from about 1 μ

l to about 10,000 μ

l.

17. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 16

wherein the step of placing the first biological sample in at least a first container further comprises the step of placing the first biological sample in at least the first container having an inner diameter in the range from about 0.02 mm to about 1.0 mm.

18. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the step of raising the temperature of the first biological sample comprises the step of adjusting the first rate.

19. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the step of lowering the temperature of the first biological sample comprises the step of adjusting the second rate.

20. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the step of holding the biological sample at a temperature comprises the step of adjusting the length of the first holding period.

21. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the step of lowering the temperature of the first biological sample comprises the step of moving ambient air past the first container at a rate at least as great as 500 meters per minute.

22. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 1

wherein the first biological sample comprises DNA and primers.

23. A method of subjecting at least a first biological sample to rapid thermal cycling, the method comprising the steps of:

(a) placing the first biological sample in at least a first container, the first container having a volume in the range from about 1 μ

l to about 10,000 μ

l;

(b) raising the temperature of the first biological sample from a first temperature to a second temperature at a first rate at least as great as 10°

C. per second, the first temperature being at least 30°

C. different than the second temperature, the temperature of the first biological sample being homogeneously raised such that no portion of the first biological sample is more than about 5°

C. warmer or cooler than any other portion of the first biological sample;

(c) holding the first biological sample at a temperature at least as great as the second temperature for not more than a first holding period, the first holding period being not greater than three seconds;

(d) lowering the temperature of the first biological sample from the second temperature to at least the first temperature at a second rate at least as great as 10°

C. per second;

(e) holding the first biological sample at a temperature at least as low as the first temperature for not more than a second holding period, the second holding period being not greater than three seconds;

(f) removing the first biological sample from the first container; and

(g) repeating steps (b) through (e) at least fifteen times.

24. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 23

further comprising the step of repeating steps (b) through (e) a plurality of times and wherein the step of raising the temperature of the first biological sample comprises the step of holding the temperature of the first biological sample at a third temperature for not more than 10 seconds, the third temperature being intermediate the first temperature and the second temperature.

25. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 23

wherein the step of placing the first biological sample in at least a first container comprises the steps of:

placing a first portion of the biological sample into the first container;

placing a second portion of the biological sample into a second container;

placing a third portion of the biological sample into a third container; and

placing a fourth portion of the biological sample into a fourth container;

and wherein the method further comprises the step of positioning the first, second, third, and fourth containers in a sample chamber and wherein the step of raising the temperature of the first biological sample comprises the step of subjecting the first, second, third, and fourth containers to infrared radiation.

26. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 23

wherein the step of raising the temperature of the first biological sample comprises the step of adjusting the first rate.

27. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 23

wherein the step of lowering the temperature of the first biological sample comprises the step of moving ambient air past the first container at a rate at least as great as 500 meters per minute.

28. A method of subjecting at least a first biological sample to rapid thermal cycling as defined in

claim 23

wherein the first biological sample comprises DNA and primers.

29. A method of subjecting at least a first biological sample containing a DNA template, an oligonucleotide primer, and a reaction buffer to perform cyclic DNA amplification using a thermostable DNA polymerase, the method comprising the steps of:

(a) placing the first biological sample in a plurality of containers, each container having a volume in the range from about 1 μ

l to about 10,000 μ

l;

(b) raising the temperature of the first biological sample placed in each of the plurality of containers from a first temperature to a second temperature at a first rate at least as great as 10°

C. per second, each of the plurality of containers being subjected to infrared radiation, the first temperature being at least 30°

C. different than the second temperature, the temperature of the first biological sample being homogeneously raising such that no portion of the first biological sample is more than about 5°

C. warmer or cooler than any other portion of the first biological sample found in the same or any of the other of the plurality of containers;

(c) holding the first biological sample at a temperature at least as great as the second temperature for not more than a first holding period, the first holding period being not greater than one second;

(d) lowering the temperature of the first biological sample from the second temperature to at least the first temperature at a second rate at least as great as 10°

C. per second by moving air past the first container at a rate at least as great as 250 meters per minute;

(e) holding the first biological sample at a temperature at least as low as the first temperature for not more than a second holding period, the second holding period being not greater than one second;

(f) removing the first biological sample from substantially all of the plurality of containers; and

(g) repeating steps (b) through (e) at least fifteen times.