Thermal cycling method

• US 8,008,046 B2
• Filed: 12/15/2008
• Issued: 08/30/2011
• Est. Priority Date: 05/17/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Goodwin, David, Maltezos, George, Johnston, Matthew, Scherer, Axel, Walker, Christopher I.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/335,413
• Publication Number:
  US 20090275014A1
• Time in Patent Office: 988 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/185 using a liquid as fluid
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/50853 with covers or lids
• 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 7/54 using spatial temperature gradients
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters

Abstract

This invention provides a method for carrying out nucleic acid amplification reactions involving heating and cooling of samples in sample vessels utilizing a heat block comprising a liquid. The method can be used to perform multiple nucleic acid amplification reactions simultaneously in which each of the reactions is performed so as to have temperature profiles. The apparatus can be used for performing PCR, and real time PCR in particular, with control and uniformity.

Claims

1. A method for performing nucleic acid amplification reactions, comprising:

a. providing at least two sample vessels, each of the at least two sample vessels comprising a reaction mixture comprising a polynucleotide sample, reagents for carrying out nucleic acid amplification, and a dye for detecting amplification;

b. cycling the temperature of the reaction mixture in the at least two sample vessels to perform multiple amplification cycles, wherein the at least two sample vessels are each in thermal contact with an oil contained within a heat block wherein the oil is actively mixed;

c. regulating the temperature of the reaction mixture in each of the sample vessels by heating and cooling a heating element in contact with a bottom surface of the heat block;

wherein the temperature between the two sample vessels has a variance of less than +/−

0.3°

C.; and

d. optically measuring the dye between or during each of the multiple amplification cycles to determine a level of amplification while the at least two sample vessels remain in thermal contact with the oil.

2. The method of claim 1, wherein the temperature between any two sample vessels has a variance of less than +/−

0.2°

C.

3. The method of claim 1, wherein the temperature between any two sample vessels has a variance of less than +/−

0.1°

C.

4. The method of claim 1 wherein the nucleic acid amplification reactions comprise PCR reactions.

5. The method of claim 4 wherein the PCR reactions comprise real time PCR (RT-PCR).

6. The method of claim 5 wherein the real time PCR reactions comprise quantitative real time PCR (QRT-PCR) to simultaneously quantify and amplify a specific portion of the polynucleotide sample.

7. The method of claim 1 wherein the multiple amplification cycles comprise either two step temperature cycles or three step temperature cycles.

8. The method of claim 1 wherein the dye for detecting the level of amplification comprise a fluorescent DNA-binding dye.

9. The method of claim 1 wherein the dye for detecting the level of amplification comprises a FRET-based probe.

10. The method of claim 1 wherein the dye for detecting the level of amplification is from a fluorescent oligonucleotide probe comprising a fluor and a quencher.

11. A method for detecting the presence of a pathogen or an infectious agent comprising performing the method of claim 1 wherein the polynucleotide sample is derived from a pathogen or an infectious agent.

12. The method of claim 11 wherein the pathogen comprises a bacteria, yeast, fungi, virus, or eukaryotic parasite.

13. The method of claim 11 wherein the infectious agent comprises influenza virus, parainfluenza virus, adenovirus, rhinovirus, coronavirus, hepatitis viruses A, B, C, D, or E, HIV, enterovirus, papillomavirus, coxsackievirus, herpes simplex virus, Epstein-Barr virus, Mycobacterium, Streptococcus, Salmonella, Shigella, Staphylcococcus, Neisseria, Pseudomonads, Clostidium, or E. coli.

14. The method of claim 1 wherein the oil is actively mixed with a pump.

15. The method of claim 1 wherein the oil is actively mixed using a stir bar.

16. The method of claim 15 wherein the stir bar is a horizontal stir bar.

17. The method of claim 15 wherein the stir bar is a fan shaped stir bar.

18. The method of claim 15 wherein the stir bar has multiple projections.

19. The method of claim 15 wherein the stir bar is magnetically responsive.

20. The method of claim 15 wherein the stir bar is mechanically connected to a motor.

21. The method of claim 1 wherein the oil is actively mixed using more than one stir bar.

22. The method of claim 1 wherein the at least two sample vessels comprise a plate.

23. The method of claim 22 wherein the number of sample vessels in the plate are 6, 12, 16, 24, 48, 96, or 384.

24. The method of claim 1 wherein the oil comprises silicone oil or carboxy-modified silicone oil, mineral oil, napthenic oil, hydroisomerized oil, or paraffinic oil.

25. The method of claim 1 wherein the oil comprises a hydrocarbon compound.