Method of Performing Digital PCR

Patent metadata

Field	Value
Publication number	US-2018327813-A1
Application number	US-201816043002-A
Country	US
Kind code	A1
Filing date	Jul 23, 2018
Priority date	Apr 19, 2012
Publication date	Nov 15, 2018
Grant date	—

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.

First claim

What is claimed is:

1 . 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.

2 . The device of claim 1 , wherein the sample holding device is a matrix.

3 . The device of claim 2 , wherein the matrix is an extracellular matrix.

4 . The device of claim 1 , wherein the sample holding device is a gel.

5 . The device of claim 4 , wherein the gel is a hydrogel.

6 . The device of claim 1 , wherein the sample holding device is located around at least a portion of the periphery of the sensor.

7 . A device for performing PCR comprising:
a sample holder configured hold a sample and to fractionate the sample into a plurality of sample volumes, wherein the sample contains an initial quantity of a target nucleic acid and wherein more than 50% of the fractions contain no more than 1 target nucleic molecule of the target nucleic acid per sample volume; a thermal cycler configured to amplify or enrich the sample to produce an amplified quantity of the target nucleic acid molecule so that an ion concentration in one or more of the sample volumes is changed; and an ion detector comprising a plurality of sensors in communication with the sample volumes, the ion detector configured to detect a change in ion concentration in the one or more of sample volumes; wherein the system is configured to determine the initial quantity of the target nucleic acid from the detected change(s) in the one or more sample volumes.

8 . The device of claim 7 , wherein the sample holding device is a matrix.

9 . The device of claim 8 , wherein the matrix is an extracellular matrix.

10 . The device of claim 7 , wherein the sample holding device is a gel.

11 . The device of claim 10 , wherein the gel is a hydrogel.

12 . The device of claim 7 , wherein the sample holding device is located around at least a portion of the periphery of the sensor.

13 . A device for performing PCR comprising:
a sample holder configured hold a sample and to fractionate the 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; a thermal cycler configured to amplify or enrich the sample to produce an amplified quantity of the target nucleic acid molecule so that an ion concentration in one or more of the sample volumes is changed; a sensing layer in chemical communication with the sample holding device; and at least one sensor in electrical communication with the sensing layer; wherein the system is configured to determine the initial quantity of the target nucleic acid from ion concentration change(s) sensed in the at least one sensor.

14 . The device of claim 13 , wherein the sample holding device is a matrix.

15 . The device of claim 14 , wherein the matrix is an extracellular matrix.

16 . The device of claim 13 , wherein the sample holding device is a gel.

17 . The device of claim 16 , wherein the gel is a hydrogel.

18 . The device of claim 13 , wherein the sample holding device is located around at least a portion of the periphery of the sensor.

Assignees

• Life Technologies Corp

Inventors

• Chen Caifu
• Mcfarland Casey
• Keys David

Classifications

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2527/119

  pH · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/6851 Primary

  Quantitative amplification · CPC title

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title