Single-molecule PCR on microparticles in water-in-oil emulsions

Patent metadata

Field	Value
Publication number	US-9260751-B2
Application number	US-201414261837-A
Country	US
Kind code	B2
Filing date	Apr 25, 2014
Priority date	Jun 19, 2006
Publication date	Feb 16, 2016
Grant date	Feb 16, 2016

Abstract

Modulation of the viscosity of the oil phase of a microemulsion used for amplification of DNA on a bead increases the homogeneity of product beads and the amount of amplified DNA per bead. Moreover the number of separate microemulsion populations that can be formed in parallel is increased using multi-well plates and mixer mill disrupter machines designed to lyse biological samples.

First claim

The invention claimed is:

1. A liquid composition comprising a plurality of microemulsions forming aqueous compartments wherein at least a portion of said aqueous compartments comprise:
a bead;
a polynucleotide template; and
oligonucleotide primers for amplifying said template;
wherein at least a portion of the oligonucleotide primers is bound to the bead, wherein said microemulsions comprise an oil phase and an aqueous phase, wherein the aqueous phase comprises from 10-30% (v/v) of the microemulsions and the oil phase comprises from 70-90% (v/v) of the microemulsions; wherein the oil phase comprises one or more low viscosity hydrocarbons with a viscosity less than 20 mPas at 25° C. in an amount from 60-85% (v/v) of the oil phase, one or more high viscosity hydrocarbons having a viscosity of greater than 20 mPas at 25° C. in an amount from 10-30% (v/v), and an emulsifier in an amount from 5-10% (v/v).

2. The liquid composition of claim 1 wherein the microemulsions comprise the high viscosity hydrocarbons in an amount from 15-25% (v/v) of the oil phase.

3. The liquid composition of claim 1 wherein the microemulsions comprise the high viscosity hydrocarbons in an amount from 17-23% (v/v) of the oil phase.

4. The liquid composition of claim 1 wherein a plurality of separate microemulsion populations are formed simultaneously in a multi-well plate using a tissue mixer mill disruptor and a metal ball in each well.

5. The liquid composition of claim 1 wherein the low viscosity hydrocarbons comprise an oxygen moiety selected from the group consisting of: a hydroxyl, an ester, an ether, and a carboxylic acid.

6. The liquid composition of claim 1 wherein the high viscosity hydrocarbons have a viscosity of 20-30 mPas at 25° C.

7. The liquid composition of claim 1 wherein the high viscosity hydrocarbons have a viscosity of 20-25 mPas at 25° C.

8. The liquid composition of claim 1 wherein the high viscosity hydrocarbons have a viscosity of 22-26 mPas at 25° C.

9. The liquid composition of claim 1 wherein the low viscosity hydrocarbons are selected from the group consisting of: 4-glyceryl isostearate, ethylene glycol, propylene glycol, cetyl propylene glycol, hexyl laurate, diethyl hexylcarbonate, and mixtures thereof.

10. The liquid composition of claim 1 wherein the low viscosity hydrocarbons have a viscosity of less than 15 mPas at 25° C.

11. The liquid composition of claim 1 wherein the low viscosity hydrocarbons have a viscosity of less than 10 mPas at 25° C.

12. The liquid composition of claim 1 wherein the low viscosity hydrocarbons have a viscosity of less than 5 mPas at 25° C.

Assignees

• Univ Johns Hopkins

Inventors

• Diehl Frank
• Kinzler Kenneth W
• Vogelstein Bert

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2565/537

  characterised by the capture oligonucleotide acting as a primer · CPC title

• C12Q2527/153

  Viscosity · CPC title