Method and apparatus for determining copy number variation using digital PCR

Patent metadata

Field	Value
Publication number	US-9487822-B2
Application number	US-17041408-A
Country	US
Kind code	B2
Filing date	Jul 9, 2008
Priority date	Mar 19, 2008
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

A method of estimating a concentration of DNA molecules in a biological sample includes storing a number of a plurality of reaction sites in a memory and distributing the biological sample among the plurality of reaction sites. The method also includes determining a number of the plurality of reaction sites characterized by a presence of one or more of the DNA molecules and computing a portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules. The method further includes estimating the concentration of the DNA molecules as a function of the portion of the plurality of reaction sites and computing a confidence interval for the estimated concentration of DNA molecules.

First claim

What is claimed is:

1. A method of distinguishing between a first sample with a first number of copies of a target gene and a second sample with a second number of copies of the target gene, the method comprising:
providing a processor;
storing a number (C) of a plurality of reaction sites in a memory;
distributing the first sample and a reference gene among reaction sites of a first test panel having the number (C) of reaction sites;
distributing the second sample and the reference gene among reaction sites of a second test panel having the number (C) of reaction sites;
performing a PCR process to amplify the first sample, the second sample, and the reference gene;
obtaining a first fluorescent image of the first test panel associated with the first sample and a first fluorescent reference image of the first test panel associated with the reference gene;
a second fluorescent image of the second test panel associated with the second sample and a second fluorescent image of the second test panel associated with the reference gene;
determining, using the first fluorescent image and the first fluorescent reference image, a first number (H 1 ) of the plurality of reaction sites characterized by a presence of one or more of the first sample;
determining, using the fluorescent second image and the second fluorescent reference image, a second number (H 2 ) of the plurality of reaction sites characterized by a presence of one or more of the second sample;
computing, using the processor, a first portion (P 1 =H 1 /C) and a second portion (P 2 =H 2 /C) of the plurality of reaction sites characterized by the presence of the one or more of the first sample and the second sample, respectively:
estimating, using the processor, the first concentration (λ 1 ) of the first sample and a second concentration (λ 2 ) of the second sample as a function of the first portion and the second portion, respectively;
computing, using the processor, a first confidence interval for the estimated concentration of the first sample and a second confidence interval for the estimated concentration of the second sample;
determining that the first confidence interval and the second confidence interval do not overlap; and
distinguishing between the first sample with the first number of copies of the target gene and the second sample with the second number of copies of the target gene as a result of the non-overlap of the first confidence interval and the second confidence interval.

2. The method of claim 1 wherein computing the first and second confidence intervals comprises:
computing, using the processor, a lower bound as a function of the first and second portions of the plurality of reaction sites and a standard deviation; and
computing, using the processor, an upper bound as a function of the first and second portions of the plurality of reaction sites and the standard deviation.

3. The method of claim 1 further comprising:
computing, for the first sample, a mean concentration ratio between the target gene and the reference gene; and
computing, for the second sample, a mean concentration ratio between the target gene and the reference gene.

4. The method of claim 3 wherein the first sample and the second sample are different from the reference gene.

5. The method of claim 1 wherein the plurality of reaction sites comprise a plurality of reaction chambers of a nanofluidic chip.

6. The method of claim 5 wherein the plurality of reaction sites comprise at least 765 reaction chambers in fluidic isolation.

7. The method of claim 1 wherein the plurality of reaction sites comprise a plurality of droplets.

8. The method of claim 1 wherein λ 1 =−ln(1−P 1 ) and λ 2 =−ln(1−P 2 ).

9. A method of estimating a ratio of a concentration of a first DNA molecule with a first number of copies of a target gene in a biological sample to a concentration of a second DNA molecule with a second number of copies of a target gene in the biological sample, the method comprising:
providing a processor;
storing a number (C) of a plurality of reaction sites in a memory;
distributing the biological sample and a reference gene among the plurality of reaction sites;
performing a PCR process to amplify the first DNA molecules, the second DNA molecules, and the reference gene;
capturing a first fluorescent image of the plurality of reaction sites associated with the first DNA molecule and a second fluorescent reference image associated with the reference gene;
capturing a second fluorescent image of the plurality of reaction sites associated with the second DNA molecule and a second fluorescent reference image associated with the reference gene;
determining, using the first fluorescent image and the first fluorescent reference image, a first number (H 1 ) of the plurality of reaction sites characterized by a presence of one or more of the first DNA molecules;
determining, using the second fluorescent image and the second fluorescent reference image, a second number (H 2 ) of the plurality of reaction sites characterized by a presence of one or more of the second DNA molecules;
computing, using the processor, a first portion (P 1 =H 1 /C) of the plurality of reaction sites characterized by the presence of the one or more first DNA molecules based on:
the stored number of the plurality of reaction sites; and
the first number of the plurality of reaction sites characterized by the presence of one or more of the DNA molecules;

computing, using the processor, a second portion (P 2 =H 2 /C) of the plurality of reaction sites characterized by the presence of the one or more second DNA molecules based on:
the stored number of the plurality of reaction sites; and
the second number of the plurality of reaction sites characterized by the presence of one or more of the DNA molecules;

estimating, using the processor, the concentration (λ 1 ) of the first DNA molecule as a function of the first portion of the plurality of reaction sites;
estimating, using the processor, the concentration (λ 2 ) of the second DNA molecule as a function of the second portion of the plurality of reaction sites;
computing a first confidence interval for the concentration of the first DNA molecule and a second confidence interval for the concentration of the second DNA molecule;
determining that the first confidence interval and the second confidence interval do not overlap; and
computing, using the processor, the ratio of the concentration of the first DNA molecule in the biological sample to the concentration of a second DNA molecule in the biological sample.

10. The method of claim 9 further comprising:
computing a mean concentration ratio between the first DNA molecule and the reference gene; and
computing a mean concentration ratio between the second DNA molecule and the reference gene.

11. The method of claim 9 wherein the plurality of reaction sites comprise a plurality of reaction chambers of a nanofluidic chip.

12. The method of claim 11 wherein the plurality of reaction sites comprise at least 765 reaction chambers in fluidic isolation.

13. The method of claim 9 wherein λ 1 =−ln(1−P 1 ).

14. The method of claim 9 wherein λ 2 =−ln(1−P 2 ).

Assignees

• Dube Simant
• Qin Jian
• Ramakrishnan Ramesh
• Fluidigm Corp

Inventors

• Dube Simant
• Qin Jian
• Ramakrishnan Ramesh

Classifications

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• G01N35/00594

  Quality control, including calibration or testing of components of the analyser · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title

• C12Q2527/137

  Concentration of a component of medium · CPC title