Emulsion systems and emulsion-based amplification of nucleic acid

Patent metadata

Field	Value
Publication number	US-9803226-B2
Application number	US-201514692827-A
Country	US
Kind code	B2
Filing date	Apr 22, 2015
Priority date	Oct 26, 2012
Publication date	Oct 31, 2017
Grant date	Oct 31, 2017

Abstract

Methods for efficient and high throughput emulsion-based nucleic amplification are provided. In some aspects, emulsion mixtures are provided that require extremely low input energy (e.g., the energy generated by pipetting) to form emulsions that are effective for nucleic acid amplification. Efficient formulations for breaking emulsions are likewise provided.

First claim

What is claimed is:

1. A method for emulsion polymerase amplification comprising:
(a) providing a mixture comprising an aqueous phase comprising polymerase reaction mixture and an oil mixture;
(b) emulsifying the mixture by passing the mixture through an orifice having a diameter between 2 mm and 50 μm with a velocity of 40 μl/s to 220 μl/s; and
(c) subjecting the emulsion to nucleic acid amplification;

wherein the oil mixture comprises mineral oil, diethylhexyl carbonate, polyglyceryl-4 isostearate, cetyl PEG/PPG-10/1-dimethicone, and hexyl laurate.

2. The method of claim 1 , wherein the mixture is passed through the orifice at least 5 times.

3. The method of claim 1 , wherein the mixture is passed through the orifice with a velocity of 60 μl/s to 180 μl/s.

4. The method of claim 1 , wherein the orifice has a diameter of between 1 mm and 100 μm.

5. The method of claim 1 , wherein the oil mixture comprises:
(a) 10-30% (w/v) mineral oil;
(b) 55-75% (w/v) diethylhexyl carbonate;
(c) 5-15% (w/v) of a composition comprising polyglyceryl-4 isostearate; cetyl PEG/PPG-10/1-dimethicone; and hexyl laurate; and
(d) 5-15% (w/v) of polyglyceryl-3-oleate or polyglyceryl-4 isostearate.

6. The method of claim 1 , further defined as a method for emulsion polymerase amplification in a plurality of reaction vessels.

7. The method of claim 6 , wherein the plurality of reaction vessels are arrayed as wells on a plate.

8. The method of claim 1 , wherein the aqueous phase comprises a plurality beads conjugated to a plurality of nucleic acid templates.

9. The method of claim 8 , wherein the beads are magnetic.

10. The method of claim 1 , further comprising:
(d) adding a breaking solution to the mixture to break the emulsion.

11. The method of claim 10 , wherein the breaking solution comprises:
(a) 20-50% 2-butanol;
(b) 20-50% 1-propanol; and
(c) 10-40% of water.

12. The method of claim 10 , wherein the breaking solution further comprises 0.1% to 2.0% SDS.

13. The method of claim 10 , wherein the breaking solution comprises:
(a) 30-50% 2-butanol;
(b) 30-50% 1-propanol; and
(c) 10-30% of water.

14. The method of claim 10 , wherein the breaking solution comprises an alkaline denaturing agent.

15. The method of claim 10 , further comprising:
(e) removing the oil or aqueous phase.

16. The method according to claim 1 , wherein the oil mixture further comprises polyglyceryl-3-oleate.

17. The method of claim 1 , wherein passing the mixture through an orifice comprises pipetting the mixture.

Assignees

• Sysmex Corp

Inventors

• Diehl Frank
• Angenendt Philipp

Classifications

• C12Q2563/159

  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 title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q2563/149

  Particles, e.g. beads · CPC title