Digital analyte analysis

• US 9,074,242 B2
• Filed: 02/11/2011
• Issued: 07/07/2015
• 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:
  US13/026,120
• Publication Number:
  US 20110250597A1
• Time in Patent Office: 1,607 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 1/1
• 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 plurality of microdroplets, each comprising a single nucleic acid template and a plurality of species of primer pairs, each species of primer pairs being specific for a different target site, wherein each of the single nucleic acid templates in the microdroplets comprise one of the different target sites that is recognized by only a single species of primer pairs, wherein the single species of the primer pairs binds to the target site of the single nucleic acid template in each microdroplet.

2. The plurality of microdroplets of claim 1, further comprising a plurality of species of probes, wherein each species hybridizes to a specific region in one of the target sites.

3. The plurality of microdroplets of claim 1, wherein the single nucleic acid template is a DNA or an RNA.

4. The plurality of microdroplets of claim 1, further comprising reagents for conducting a polymerase chain reaction.

5. The plurality of microdroplets of claim 2, wherein the species of the probes contain a detectable label.

6. The plurality of microdroplets of claim 5, wherein one of the species of the probes comprise two groups of the probes that hybridize to the specific region, wherein each group comprises a different detectable label.

7. The plurality of microdroplets of claim 5, wherein one of the species of the probes comprise two groups of the probes that hybridize to the specific region, wherein a first group comprises the detectable label and a second group does not include the detectable label.

8. The plurality of microdroplets of claim 5 wherein each species of the probes comprise a different detectable label.

9. The plurality of microdroplets of claim 5, wherein the detectable label is a fluorescent label.