Method for rapid thermal cycling of biological samples

• US 6,787,338 B2
• Filed: 08/11/1998
• Issued: 09/07/2004
• Est. Priority Date: 06/04/1990
• Status: Expired due to Fees

• Current Assignee: University of Utah (State of Utah)
• Original Assignee: University of Utah (State of Utah)
• Inventors: Hillyard, David R., Wittwer, Carl T.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/132,156
• Publication Number:
  US 20010007759A1
• Time in Patent Office: 2,219 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/91.2
• 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 a sample to rapid thermal cycling, said method comprising:

a) contacting a sample holder containing a sample with heated fluid, thereby raising the temperature of the sample to a first temperature, and holding the sample at about said first temperature for a first predetermined period of time;

b) contacting the sample holder with non-heated fluid, thereby lowering the temperature of the sample to a second temperature, an holding the sample at about said second temperature for a second predetermined period of time;

c) contacting the sample holder with heated fluid, thereby raising the temperature of the sample to a third temperature, and holding the sample at about said third temperature for a third predetermined period of time;

wherein steps a) through c) are completed within a time range of about 30 seconds to 60 seconds and repeated at least one time; and

wherein said sample holder has a thermal mass which provides for completing said cycle within said time range.

2. The method of claim 1, wherein said sample holder is one of a plurality of sample holders.

3. The method of claim 1, wherein the first predetermined period of time is about 8 seconds or less.

4. The method of claim 3, wherein the first predetermined period of time is less than 1 second.

5. The method of claim 1, wherein said lowering in step b) occurs in about 25 seconds or less.

6. The method or claim 1, wherein said lowering in step b) occurs at an average rate of between at least about 1.5°

C. per second and upto about 4.1°

C. per second.

7. The method of claim 1, wherein the difference between said first and said second temperature is up about 42°

C.

8. The method of claim 1, wherein the second predetermined period of time is about 5 seconds or less.

9. The method claim 8, wherein the second predetermined period of time is less than 1 second.

10. A method of amplifying a nucleic acid, said method comprising:

a) heating a sample holder containing a sample comprising a nucleic acid and amplification primer, thereby raising the temperature of the sample to a first temperature, and holding the sample at about said first temperature for a first predetermined period of time to denature said double-stranded nucleic acid;

b) cooling the sample holder, thereby lowering the temperature of the nucleic acid sample to a second temperature, and holding the sample at about said second temperature for a second predetermined period of time to anneal said amplification primer;

c) heating the sample holder, there raising the temperature of the nucleic acid sample to a third temperature, and holding the sample at about said third temperature for a third predetermined period of time to allow amplification of said nucleic acid;

d) repeating steps a) through c), wherein steps a) through c) are completed within a time range of about 30 seconds to 60 seconds;

wherein said sample holder has a thermal mass which provides for completing said cycle within said time range.

11. The method of claim 10, wherein said sample further comprises a nucleic acid polymerizing enzyme.

12. The method of claim 10, wherein said sample holder is one of a plurality of sample holders.

13. The method of claim 10, wherein the first predetermined period of time is less than 1 second.

14. The method of claim 10, wherein said lowering in step b) occurs in about 25 seconds or less.

15. The method of claim 14, wherein said lowering in step b) occurs in about 9 seconds.

16. The method of claim 10, wherein said lowering in step b) occurs at an average rate of between at least about 1.5°

C. per second and up to about 4.1°

C. per second.

17. The method of claim 10, wherein the difference between said first and said second temperature is up to bout 42°

C.

18. The method of claim 10, wherein the second predetermined period of time is about 5 seconds or less.

19. The method of claim 18, wherein the second predetermined period of time is less than 1 second.

20. A method of subjecting a nucleic acid sample to rapid thermal cycling to amplify a nucleic acid, said method comprising:

a) raising the temperature of a sample comprising a nucleic acid to a first temperature and holding the sample at about said first temperature for a first predetermined period of time;

b) lowering the temperature of the sample to a second temperature at a rate at least about 1.5°

C. per second and holding the sample at about said second temperature for a second predetermined period of time;

c) repeating steps a) and b) at least once in order to amplify said nucleic acid, wherein each cycle of steps is completed within a time range of about 30 seconds to 60 seconds.

21. The method of claim 20, further comprising;

an intervening step d) between step b) and step c) wherein said step comprises raising the temperature of the sample to a third temperature intermediate to said first and second temperatures and holding the sample at about said third temperature for a third predetermined period of time wherein said steps a), b) and d) are completed within said time range.

22. The method of claim 20 or 21, wherein said lowering in step b) is at a rate of up to about 4.1°

C. per second.

23. The method of claim 20 or 21, wherein the difference between said first and said second temperature is up to about 42°

C.

24. The method of claim 20 or 21, wherein the first predetermined period of time is about 8 seconds or less.

25. The method of claim 20 or 21, wherein the first predetermined of time is less than 1 second.

26. The method of claim 20 or 21, wherein the second predetermined period of time is about 20 seconds or less.

27. The method of claim 20 or 21, wherein the second predetermined period of time is less than 1second.

28. The method of claim 20 or 21, further comprising the step;

e) detecting the amplification of the nucleic acid by fluorescence.

29. A method of subjecting a sample to rapid thermal cycling, said method comprising:

a) heating a sample holder containing a sample with heated fluid, thereby raising the temperature of the sample to a first temperature, and holding the sample at about said first temperature for a first predetermined period of time;

b) cooling the sample holder with non-heated fluid, thereby lowering the temperature of the sample to a second temperature, and holding the sample at about said second temperature for a second predetermined period of time;

c) heating the sample holder with heated fluid, thereby raising the temperature of the sample to a third temperature, and holding the sample at about said third temperature for a third predetermined period of time;

wherein steps a) through c) are completed within a time range of about 30 seconds to 60 seconds and repeated at least one time; and

wherein said sample holder has thermal mass which provides for completing said cycle within said time range.

30. The method of claim 29, wherein said sample holder is one of a plurality of sample holders.

31. The method of claim 29, wherein the first predetermined period of time is less than 1 second.

32. The method of claim 29, wherein said lowering an step b) occurs in about 25 seconds or less.

33. The method of claim 29, wherein said lowering in step b) occurs in about 9 seconds.

34. The method of claim 29, wherein said lowering in step b) occurs at an average rate of between at least about 1.5°

C. per second and up to about 4.1°

C per second.

35. The method of claim 29, wherein the difference berween said first and said second temperature is up to about 42°

C.

36. The method of claim 29, wherein the second predetermined period of time is about 5 seconds or less.

37. The method of claim 29, wherein the second predetermined period of time is less than 1 second.

38. The method of claim 29, wherein said sample comprises a nucleic acid.

39. The method of claim 38, wherein said sample further comprises a nucleic acid polymerizing enzyme.