Digital analyte analysis

• US 9,228,229 B2
• Filed: 03/12/2013
• Issued: 01/05/2016
• 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: Olson, Jeffrey, Link, Darren R.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US13/796,901
• Publication Number:
  US 20130224751A1
• Time in Patent Office: 1,029 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 method for analyzing a nucleic acid template in a biological sample, the method comprising the steps of:

forming a plurality of microdroplets, each comprising a nucleic acid template and a plurality of sets of primer pairs, each set of primer pairs being specific for a different target site on a different nucleic acid template, binding only a single primer pair to the target site on the nucleic acid template in each microdroplet;

amplifying a single target site from the nucleic acid template in each of the microdroplets using one of the sets of primer pairs specific for the single target site to generate amplification products;

attaching a sequencing adaptor and a barcode sequence to the amplification products; and

sequencing the amplification products, thereby to analyze the nucleic acid template.

2. The method according to claim 1, wherein the amplification products are attached to a bead.

3. The method according to claim 2, wherein the amplification products are attached to a surface.

4. The method according to claim 3, wherein the surface is a surface of a flow cell.

5. The method according to claim 3, wherein the amplification products are covalently attached to the surface.

6. The method according to claim 1, wherein sequencing is sequencing-by-synthesis.

7. The method according to claim 6, wherein sequencing-by-synthesis is single-molecule sequencing-by-synthesis.

8. The method according to claim 1, wherein amplifying is by the polymerase chain reaction.

9. The method according to claim 1, wherein the single nucleic acid template is a DNA or an RNA.

10. The method according to claim 1, wherein the nucleic acid template is associated with a cancer.