Method of performing digital PCR

• US 10,030,262 B2
• Filed: 04/19/2013
• Issued: 07/24/2018
• 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
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/395,456
• Publication Number:
  US 20150099644A1
• Time in Patent Office: 1,922 Days
• Field of Search: 435 61, 435 912
• 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:

providing a sample comprising an initial quantity of a target nucleic acid molecule;

amplifying or enriching the sample to produce an amplified quantity of the target nucleic acid molecule;

after amplifying, fractionating the sample into a plurality of sample volumes wherein more than 50% of the fractions contain no more than 1 of the target nucleic acid molecules;

changing the ion concentration in one or more of the sample volumes by subjecting the plurality of sample volumes to conditions for amplification;

detecting the change in ion concentration in the one or more of sample volumes; and

determining the initial 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.

14. The method of claim 1 where the change in ion concentration is an increase.

16. The method of claim 1 further comprising covering the sample volumes with a cover or lid, the cover or lid comprising a heating element configured to provide conditions for amplification.

17. The method of claim 1 wherein the sample volumes are contained in or on a sample holding device comprising a matrix, the matrix comprising a gel or a polymer forming a continuous layer over a surface of the sample holding device.

18. The method of claim 1 further comprising positioning each of the plurality of samples in a plurality of isolated positions, wherein:

the plurality of isolated position are in chemical communication with a plurality of sensors; and

two or more of the plurality of isolated position are in chemical communication with a single sensor of the plurality of sensors.

19. The method of claim 1 further comprising:

prior to fractionating the sample, generating an amplicon with two target specific PCR primers;

supplying the sample with polymerase and a plurality of dNTPs; and

after fractionating the sample, detecting hydrogen ions.

20. The method of claim 19 wherein the plurality of dNTPs consist of only three different types of dPTPs.

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%;

changing a concentration of ions in two or more of the sample volumes by subjecting the plurality of sample volumes to at least one amplification cycle;

detecting the change in ion concentration in the two or more of sample volumes after the at least one amplification cycle;

quantitating an initial amount of target nucleic acid;

wherein detecting comprises detecting both positive and negative ions.

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

providing a sample comprising an initial quantity of a target nucleic acid molecule;

fractionating the sample into a plurality of wells so that more than 50% of the wells contain no more than one of target nucleic acid molecules, wherein at least one of the wells contains at least one of the target nucleic acid molecule, at least one of the wells contain a portion of the sample with no target nucleic acid molecule, and at least one of the wells contains no sample and no target nucleic acid molecule;

changing the ion concentration in one or more of the sample volumes by subjecting the plurality of sample volumes to conditions for amplification;

detecting the change in ion concentration in the one or more of sample volumes;

determining the initial quantity of the target nucleic acid in the sample; and

detecting which well or wells contain no sample and no target nucleic acid molecule.