Rapid thermocycling for sample analysis

• US 6,210,882 B1
• Filed: 01/29/1998
• Issued: 04/03/2001
• Est. Priority Date: 01/29/1998
• Status: Expired due to Term

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic), University of Pittsburgh of The Commonwealth System of Higher Education
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic), University of Pittsburgh of The Commonwealth System of Higher Education
• Inventors: Huhmer, Andreas, Landers, James P, Oda, Robert P., Craighead, James R.
• Primary Examiner(s): Redding, David A.
• Application Number:
  US09/015,278
• Time in Patent Office: 1,160 Days
• Field of Search: 435/6, 435/91.2, 435/69.1, 435/287.2, 436/174, 436/89, 165/61, 165/101 FOR
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/185 using a liquid as fluid
• CPC Class Codes: B01L 2300/1872 Infrared light
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• 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: F16K 13/10 by means of liquid or granular medium
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

Methods for performing rapid and accurate thermocycling on a sample are disclosed. Use of non-contact heating and cooling sources allows precise temperature control with sharp transitions from one temperature to another to be achieved. A wide range of temperatures can be accomplished according to these methods. In addition, thermocycling can be performed without substantial temperature gradients occurring in the sample. Apparatus for achieving these methods are also disclosed. A method for pumping a sample through microchannels on a microchip using a non-contact heat source is also disclosed.

Claims

1. A method for thermocycling a sample comprising:

(a) placing a volume of sample in the nanoliter range in a reaction vessel having a large surface to volume ratio;

(b) heating the sample to a first desired temperature using the optical energy of a non-contact heat source that directly heats the sample itself at a rate of between about 10°

C./second and 100°

C./second;

(c) positively cooling the sample to a second desired temperature by a non-contact cooling source below ambient temperature directed at the reaction vessel at a rate of between about 5°

C./second and 50°

C./second; and

(d) sequentially repeating steps (b) and (c) to perform a desired number of thermocycles wherein each thermocycle is completed in between about 0.3 seconds and about 20 seconds.

2. The method of claim 1, further including at least one intermediate heating step to achieve at least one intermediate temperature lower than said first desired temperature.

3. The method of claim 1, further including at least one intermediate cooling step to achieve at least one intermediate temperature higher than said second desired temperature.

4. The method of claim 1, wherein each desired temperature is maintained for a desired dwell time.

5. The method of claim 4, wherein said desired dwell times range from milliseconds to about 20 seconds.

6. The method of claim 1, wherein the sample is sequentially:

(a) heated to a first desired temperature and maintained at that temperature for a desired dwell time;

(b) cooled to a second desired temperature and maintained at the second desired temperature for a desired dwell time; and

(c) heated to a third desired temperature and maintained at the third desired temperature for a desired dwell time;

wherein the third desired temperature is higher than the second desired temperature, but lower than the first desired temperature; and

wherein steps (a), (b) and (c) are sequentially repeated to perform the desired number of thermocycles.

7. The method of claim 6, wherein said first desired temperature is between about 90 and 98°

C., said second desired temperature is between about 40 and 75°

C., and said third desired temperature is between about 60 and 80°

C.

8. The method of claim 7, wherein the dwell time at said first desired temperature is between about 1 to 20 seconds, the dwell time at said second desired temperature is between about 1 to 20 seconds, and the dwell time at said third desired temperature is between about 1 to 20 seconds.

9. The method of claim 1, wherein the suitable reaction vessel is selected from the group consisting of a capillary tube, a microchip, a microchamber and a microtiter plate.

10. The method of claim 1, wherein heating is effected by use of an IR source.

11. The method of claim 10, wherein said IR source is a halogen lamp.

12. The method of claim 10, wherein said IR source is a tungsten lamp.

13. The method of claim 1, wherein cooling is effected by use of a compressed air source.

14. The method of claim 13, wherein the pressure of the air from said compressed air source is between about 1 and 150 psi.

15. The method of claim 1, wherein between about 5 and 100 thermocycles are carried out on the sample.

16. The method of claim 1, wherein said method is used to amplify DNA in said sample.

17. The method of claim 1, wherein a microprocessor is used to control said heating and cooling steps.

18. The method of claim 17, wherein said first desired temperature is less than or equal to about 120°

C. and said second desired temperature is greater than or equal to about 4°

C.

19. The method of claim 1, wherein said method is used to effect protein folding and unfolding.

20. The method of claim 1, wherein said method is used to effect activation of an enzyme.

21. The method of claim 1, wherein said method is used to effect sequencing of a protein or peptide.

22. The method of claim 2, further including at least one intermediate cooling step to achieve at least one intermediate temperature higher than said second desired temperature.

23. The method of claim 1, wherein said method is used to effect denaturation of enzymes.