THERMAL REACTION DEVICE AND METHOD FOR USING THE SAME

• US 20090291435A1
• Filed: 03/20/2006
• Published: 11/26/2009
• Est. Priority Date: 03/18/2005
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated, Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Facer, Geoffrey, Unger, Marc A., McBride, Lincoln
• Granted Patent: US 8,828,663 B2
• Time in Patent Office: Days
• US Class Current: 435/6.19
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• 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: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/453 Cells therefor

Abstract

Devices and methods for performing the relative concentration of a target in a sample, the sample containing both target and non-target components, the method performed by partitioning the sample into a large number of reaction volumes such that the target is concentrated relative to the non-target, and performing a detection assay upon each reaction volume to detect the target.

Claims

1. A method for detecting the presence and/or quantity of a target polynucleotide in a sample, the method comprising:

(a) providing a microfluidic device having at least one sample channel and a plurality of valves distributed along the length of the sample channel, wherein the sample channel is adapted to be partitioned into a plurality of separate reaction chambers by the closing of one or more valves,

(b) introducing a sample having at least one amplifiable target into the sample channel,

(c) closing the plurality of valves to form separate reaction chambers so that the amplifiable target is isolated into at least one chamber,

(d) conducting a polynucleotide amplification reaction,

(e) detecting the presence of a polynucleotide amplification product in at least one reaction chamber to determine the presence and/or quantity of the target polynucleotide in the sample.

2. The method of claim 1 wherein the polynucleotide amplification reaction is real-time PCR.

3. The method of claim 1 in which closing the plurality of valves results in the formation of at least 1000 separate reaction chambers.

4. The method of claim 1 in which closing the plurality of valves results in the formation of at least 5000 separate reaction chambers.

5. The method of claim 1 in which closing the plurality of valves results in the formation of at least 10000 separate reaction chambers.

6. The method of claim 1 wherein the sample additionally comprises a polymerase enzyme.

7. The method of claim 1 wherein the sample additionally comprises a primer that binds specifically to the target polynucleotide.

8. The method of claim 1 wherein the sample additionally comprises a fluorescent probe.

9. The method of claim 8 wherein the sample additionally comprises a second fluorescent probe.

10. The method of claim 7 wherein the sample additionally comprises a second primer that does not bind specifically to the target polynucleotide, but binds specifically to another polynucleotide.

11. The method of claim 7 wherein the target polynucleotide is a mutant polynucleotide having a single nucleotide substitution, addition or deletion.

12. The method of claim 10 wherein the first primer binds specifically to a mutant polynucleotide and the second primer binds specifically to the wild-type polynucleotide, thereby reducing the number of wild-type polynucleotides available for non-specific binding, thereby increasing specificity of the method.

13. The method of claim 1 further comprising absolute quantification of a target polynucleotide.

14. The method of claim 1 wherein the target polynucleotide is a mutant polynucleotide, and wherein the sensitivity of detecting the mutant polynucleotide is such that the mutant polynucleotide may be detected in a sample having a mutant:

wild-type ratio of at least 1;

1×

10³.

15. The method of claim 14 wherein the sensitivity of detecting the mutant polynucleotide is such that the mutant polynucleotide may be detected in a sample having a mutant:

wild-type ratio of at least 1;

1×

10⁵.

16. The method of claim 1 wherein the sample channel has at least one dimension of between 10 and 1000 μ

m.

17. The method of claim 1 wherein the sample channel has at least one dimension of between 50 and 500 μ

m.

18. A system for performing massive partitioning of a sample, PCR, and detection of a PCR product, the system comprising a microfluidic device having at least one flow channel and a plurality of valves distributed along the length of the flow channel, wherein the flow channel is adapted to be partitioned into a plurality of isolated reaction chambers upon actuation of the valves;

and a heat-exchange plate in thermal contact with the microfluidic device.

19. The system of claim 18 further comprising a detector for detecting the presence of a PCR product.

20. The system of claim 18 wherein the flow channel is adapted to be partitioned into at least 1000 isolated reaction chambers.

21. A device adapted for detection of a target in a sample by performing massive partitioning of a sample, PCR, and detection of a PCR product, the device comprising a microfluidic device having at least one flow channel and a plurality of elastomeric valves distributed along the length of the flow channel, wherein the flow channel is partitioned into at least 1000 isolated reaction chambers upon actuation of the elastomeric valves.

22. The device of claim 21 wherein the sample channel has at least one dimension of between 10 and 1000 μ

m.

23. The device of claim 21 wherein the device is capable of detecting a target polynucleotide in a sample having a target:

wild-type ratio of at least 1;

1×

10⁵.

24. The use of a microfluidic device having at least one flow channel and a plurality of elastomeric valves dispersed along the length of the flow channel to perform massive partitioning of a sample, PCR, and detection of a PCR product.

25. The use of claim 24 wherein the PCR product is an amplified sequence of a polynucleotide whose expression is associated with a disease or with drug resistance.

26. The use of claim 25 wherein the polynucleotide is part of a gene within the ras oncogene family.

27. The use of claim 25 wherein the polynucleotide is part of a tyrosine kinase gene.