System for hot-start amplification via a multiple emulsion

• US 8,709,762 B2
• Filed: 03/02/2011
• Issued: 04/29/2014
• Est. Priority Date: 03/02/2010
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hindson, Benjamin J.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): OYEYEMI, OLAYINKA A
• Application Number:
  US13/039,233
• Publication Number:
  US 20110217736A1
• Time in Patent Office: 1,154 Days
• Field of Search: 435/91.2, 435/194
• US Class Current: 435/91.2
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12N 9/12 transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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

Abstract

System, including methods, apparatus, compositions, and kits, for making and using compound droplets of a multiple emulsion to supply an amplification reagent, such as a heat-stable DNA polymerase or DNA ligase, to an aqueous phase in which the compound droplets are disposed. The compound droplets may be induced to supply the amplification reagent by heating the multiple emulsion, to achieve hot-start amplification.

Claims

1. A method of nucleic acid amplification, comprising providing a composition comprising a plurality of compound droplets disposed in an aqueous phase, wherein each compound droplet comprise a plurality of aqueous droplets, each aqueous droplet enclosed by an oil droplet and each aqueous droplet containing at least one amplification reagent;

heating the composition to cause fusion of the aqueous droplets with the aqueous phase, such that the at least one amplification reagent is added to the aqueous phase to form at least one reaction mixture; and

cycling the at least one reaction mixture thermally to encourage amplification of a nucleic acid target.

2. The method of claim 1, further comprising a step of detecting one or more signals corresponding to amplification of the nucleic acid target in the at least one reaction mixture.

3. The method of claim 1, wherein the aqueous droplets contain DNA polymerase.

4. The method of claim 3, wherein the DNA polymerase is a Taq DNA polymerase.

5. The method of claim 1, wherein the at least one amplification reagent includes magnesium, at least one deoxynucleotide triphosphate, at least one primer, or a combination thereof.

6. The method of claim 1, wherein the aqueous phase is continuous.

7. The method of claim 1, wherein the aqueous phase is dispersed.

8. The method of claim 1,

wherein the aqueous phase lacks an effective amount of at least one PCR reagent that is contained by the aqueous droplets, wherein;

the at least one reaction mixture is capable of PCR amplification of a nucleic acid target, if present, in the reaction mixture, and

wherein cycling the at least one reaction mixture thermally encourages PCR amplification of the nucleic acid target.

9. The method of claim 1, wherein fusion of the aqueous droplets with the aqueous phase occurs at least predominantly after the composition has been heated to a temperature of at least about 50°

C.

10. The method of claim 8, wherein the aqueous phase in the composition provided includes an effective amount of each reaction component for the PCR amplification except the at least one PCR reagent.

11. A method of nucleic acid amplification, comprising providing a composition comprising a plurality of compound droplets disposed in an aqueous phase, wherein each compound droplet comprise a plurality of aqueous droplets, each aqueous droplet enclosed by an oil droplet and each aqueous droplet containing a heat stable polymerase;

heating the composition such that the aqueous droplets with the aqueous phase, to form at least one reaction mixture capable of PCR amplification of a nucleic acid target, if present, in the at least one reaction mixture; and

cycling the at least one reaction mixture thermally to encourage PCR amplification.

12. A composition for nucleic acid amplification, comprising an aqueous phase that is dispersed or continuous;

a plurality of compound droplet disposed in the aqueous phase;

wherein each compound droplet comprise a plurality of aqueous droplets, each aqueous droplet enclosed by an oil droplet and each aqueous droplet containing at least one amplification reagent, wherein heating the composition causes the aqueous droplets to fuse with the aqueous phase to supply the at least one amplification reagent to the aqueous phase.

13. The composition of claim 12, wherein the aqueous droplets contains a heat-stable DNA polymerase.

14. The composition of claim 12, where heating the composition forms at least one reaction mixture capable of amplification of a nucleic acid target, if present, in the reaction mixture.