Method of Performing Digital PCR

• US 20150099644A1
• Filed: 04/19/2013
• Published: 04/09/2015
• Est. Priority Date: 04/19/2012
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Caifu, McFarland, Casey, Keys, David
• Granted Patent: US 10,030,262 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

A method of detection of a target nucleic acid is provided. The method includes fractionating a sample into a plurality of sample volumes wherein more than 50% of the fractions contain no more than 1 target nucleic acid molecule per sample volumes, and subjecting the plurality of sample volumes to conditions for amplification. The method further includes detecting a change in ion concentration in a sample volume wherein a target nucleic acid is present, counting the number of fractions with an amplified target nucleic acid, and determining the quantity of target nucleic acid in the sample.

Claims

1. A method of detection of a target nucleic acid comprising:

fractionating a sample into a plurality of sample volumes wherein more than 50% of the fractions contain no more than 1 target nucleic acid molecule per sample volumes;

subjecting the plurality of sample volumes to conditions for amplification;

detecting a change in ion concentration in a sample volume wherein a target nucleic acid is present;

counting the number of fractions with an amplified target nucleic acid; and

determining the quantity of target nucleic acid in the sample.

2. The method of claim 1 further comprising combining a sample with primers and probes for amplification.

3. The method of claim 1 wherein the change in ion concentration is an increase in ion concentration.

4. The method of claim 1 further comprising combining a sample with a bead.

5. The method of claim 1 further comprising loading the sample on a substrate wherein the substrate includes at least one well.

6. The method of claim 5 further comprising sealing the at least one well with an immiscible fluid layer

7. The method of claim 5 wherein the at least one well includes a coating.

8. The method of claim 7 wherein the coating is a hydrophilic coating.

9. The method of claim 1 further comprising positioning each of the plurality of samples in a plurality of isolated positions, wherein each of the plurality of isolated position is in chemical communication with a sensor.

10. The method of claim 1 wherein the change in ion concentration in an increase in hydrogen ions.

11. The method of claim 1 wherein the change in ion concentration is a change in pH.

12. The method of claim 1 further comprising quantifying the amount of a target nucleic acid.

13. A method for performing absolute quantification of a nucleic acid comprising:

diluting a sample containing an initial amount of a target nucleic acid into a plurality of sample volumes wherein the percentage of reaction areas containing one or more target nucleic acid molecules is greater than 50% and less than 100%;

subjecting the plurality of sample volumes to at least one amplification cycle;

detecting a change in ion concentration in at least one of the plurality of sample volumes as a result of the at least one amplification cycle;

quantitating an initial amount of target nucleic acid.

14. The method of claim 13 where the change in ion concentration is an increase.

15. The method of claim 13 wherein the ion is a positive ion.

16. The method of claim 15 wherein the positive ion is hydrogen ion.

17. The method of claim 13 wherein the ion is a negative ion.

18. A device for performing PCR comprising a sample holder configured to fractionate a sample into a plurality of sample volumes wherein more than 50% of the fractions contain no more than 1 target nucleic molecules per sample volume and further configured to hold a sample;

a sensing layer in chemical communication with the sample holding device; and

at least one sensor in electrical communication with the sensing layer.

19. The device of claim 18 wherein the sample holding device is a matrix.

20. The device of claim 19 wherein the matrix is an extracellular matrix.

21. The device of claim 18 wherein the sample holding device is a gel.

22. The device of claim 21 wherein the gel is a hydrogel.

23. The device of claim 18 wherein the sample holding device is located around at least a portion of the periphery of the sensor.