Thermal reaction device and method for using the same

• US 8,828,663 B2
• Filed: 03/20/2006
• Issued: 09/09/2014
• 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: Fluidigm Corporation, Regents of the University of California (University of California)
• Inventors: Unger, Marc, McBride, Lincoln, Facer, Geoffrey, Lucero, Michael, Nassef, Hany Ramez
• Primary Examiner(s): Kim, Young J
• Application Number:
  US11/912,502
• Publication Number:
  US 20090291435A1
• Time in Patent Office: 3,095 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 highly sensitive and specific detection of mutant alleles in a high background of wild-type alleles, comprising

a) obtaining an assay composition comprising first polynucleotides encoding a mutant allele of a gene and second polynucleotides encoding a wild-type allele of the gene, wherein the ratio of first polynucleotides to second polynucleotides is 1:

1×

10³ or lower;

b) distributing the assay composition, or a portion thereof, into at least 10,000 aliquots, thereby producing a subset of aliquots in which the ratio of first polynucleotides to the second polynucleotides is greater than the ratio of first polynucleotides to second polynucleotides in said assay composition, wherein some aliquots comprise a first polynucleotide and essentially all aliquots comprise at least one second polynucleotide;

c) combining the assay composition with amplification reagents prior to step (b) or combining the at least 10,000 aliquots with amplification reagents after step (b), wherein said amplification reagents;

i) comprise amplification primers specific for amplifying at least a portion of the mutant allele, and

ii) do not comprise amplification primers specific for amplifying the wild-type allele;

d) amplifying the at least a portion of the mutant allele encoded in the first polynucleotides; and

e) detecting the presence or absence of amplification in each aliquot.

2. The method of claim 1 wherein the first and second polynucleotides are derived from the genome of the same individual.

3. The method of claim 1 wherein said assay composition is combined with all of said amplification reagents prior to step (b).

4. The method of claim 1 wherein the mutant allele differs from the wild-type allele by one or more point mutations.

5. The method of claim 1 wherein the mutant allele differs from the wild-type allele by an insertion or deletion.

6. The method of claim 1 wherein the amplification in (d) is by real-time PCR.

7. The method of claim 1 wherein the amplification mixture additionally comprises a nonextendible primer that binds specifically to the wild-type polynucleotide.

8. The method of claim 1 wherein the ratio of first polynucleotides to second polynucleotides is 1:

1×

10⁵ or lower.

9. The method of claim 1, wherein the presence of amplification is determined by detecting a fluorescent signal.

10. The method of claim 1 wherein the gene is an oncogene.

11. The method of claim 1 wherein the assay composition is combined with amplification reagents prior to step (b).

12. The method of claim 1 wherein some aliquots comprise a first polynucleotide and essentially all aliquots comprise at least a plurality of second polynucleotides.

13. The method of claim 12 wherein some aliquots comprise a first polynucleotide and essentially all aliquots comprise hundreds of second polynucleotides.