Digital analyte analysis

• US 8,535,889 B2
• Filed: 02/11/2011
• Issued: 09/17/2013
• Est. Priority Date: 02/12/2010
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Raindance Technologies Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Larson, Jonathan William, Zhong, Qun, Link, Darren Roy
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US13/026,107
• Publication Number:
  US 20110244455A1
• Time in Patent Office: 949 Days
• Field of Search: 435/6.12, 435/91.1, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• 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 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, methods of the invention involve forming sample droplets containing, on average, a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons.

Claims

1. A method for analyzing a target nucleic acid, the method comprising the steps of:

forming droplets containing a single target nucleic acid and one or more amplification reagents;

amplifying the target in the droplets;

distinguishing droplets containing amplicon from the target and amplicon from a variant of the target generated by polymerase error by using two differently labeled-hybridization probes, one hybridizing to the target and one hybridizing to a specific variant of the target; and

analyzing target amplicons.

2. The method according to claim 1, wherein said amplifying step is a polymerase chain reaction and the one or more amplification reagents includes one or more primer pairs.

3. The method according to claim 1, wherein said distinguishing step comprises flowing said droplets in a microfluidic channel.

4. The method according to claim 1, wherein said analyzing step comprises detecting said amplicons by hybridization to detectably-labeled probes.

5. The method according to claim 1, wherein said analyzing step is conducted on amplicon from droplets that were not distinguished in said distinguishing step.

6. The method according to claim 1, wherein said forming step comprises:

flowing a stream of first sample fluid comprising nucleic acids such that it intersects two opposing streams of flowing carrier fluid, wherein said carrier fluid is immiscible with said sample fluid, thereby forming a plurality of first droplets comprising the first sample fluid; and

merging each of the plurality of first droplets comprising the first sample fluid with a portion of a second fluid comprising one or more amplification reagents, wherein the portion of a

second fluid optionally is a droplet.

7. The method according to claim 6, wherein said carrier fluid is oil.

8. The method according to claim 7, wherein said oil comprises a surfactant.

9. The method according to claim 8, wherein the surfactant is a fluorosurfactant.

10. The method according to claim 1, wherein said analyzing step comprises:

determining a number of droplets that contain only wild-type target;

determining a number of droplets that contain only a variant of the target.

11. The method according to claim 10, wherein presence of droplets containing only said variant is indicative of a disease.

12. The method according to claim 11, wherein the disease is cancer.

13. The method according to claim 10, wherein the variant is an allelic variant.

14. The method according to claim 13, wherein the allelic variant is a single nucleotide polymorphism.