Emulsion compositions

• US 7,622,280 B2
• Filed: 06/11/2004
• Issued: 11/24/2009
• Est. Priority Date: 11/16/2001
• Status: Expired due to Term

• Current Assignee: UK Research and Innovation
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Holliger, Phillip, Ghadessy, Farid
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US10/866,392
• Publication Number:
  US 20050064460A1
• Time in Patent Office: 1,992 Days
• Field of Search: 435/5, 435/6, 435/320.1, 435/69.1, 536/23.1, 424/450
• US Class Current: 435/91.2
• CPC Class Codes: A61K 47/24 containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 47/44 Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin synthetic glycerides, e.g. medium-chain triglycerides, A61K47/14
• CPC Class Codes: A61K 9/107 Emulsions ; Emulsion preconcentrates; Micelles composition of emulsions A61K47/00
• CPC Class Codes: A61K 9/1676 having a drug-free core with discrete complete coating layer containing drug adsorbates of liquid drug formulations on inert powders without simultaneous granulation step A61K9/141; with further drug-free outer coating A61K9/5078; drug conjugated to non-active particles A61K47/6921
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

An emulsion is useful in allowing a wide variety of gene products to be expressed via eukaryotic in vitro expression. The emulsion comprises a silicone based surfactant, a hydrophobic phase and a hydrophilic phase; wherein the hydrophilic phase comprises a plurality of compartments containing a functional in vitro eukaryotic expression system.

Claims

1. A method for increasing the concentration of a nucleic acid molecule comprising the steps of:

(a) forming microcapsules from a water-in-oil emulsion consisting of;

a chemically inert silicone-based surfactant, a hydrophobic oil external phase, and a hydrophilic aqueous internal phase comprising the microcapsules, wherein the microcapsules include a nucleic acid molecule, a bead capable of being linked to the nucleic acid molecule, and an aqueous solution comprising components necessary to perform nucleic acid amplification;

(b) amplifying the nucleic acid molecule in the microcapsules to form amplified copies of the nucleic acid molecule, wherein the amplification employs a thermal cycling process, thereby increasing the concentration of the nucleic acid molecule in the microcapsules.

2. The method of claim 1 wherein the amplified copies are captured to the beads in the microcapsules.

3. The method of claim 1 wherein the nucleic acid amplification is performed using a method selected from the group consisting of Qb replicase amplification, ligase chain reaction, self sustained sequence replication, and strand displacement amplification.

4. The method of claim 1 wherein the water-in-oil emulsion comprises a water to oil ratio of 1 to 2.5.

5. The method of claim 1 wherein the nucleic acid molecule is genomic DNA or cDNA.

6. The method of claim 1 wherein the nucleic acid molecule comprises a tag selected from the group consisting of biotin, digoxigenin, and 2,4-dinitrophenyl.

7. The method of claim 1 wherein the bead comprises a coating selected from the group consisting of avidin, streptavidin, anti-digoxigenin antibodies, and anti-2,4-dinitrophenyl antibodies.

8. The method of claim 1 wherein the bead is selected from the group consisting of polystyrene and magnetic beads.

9. The method of claim 1 wherein the chemically inert silicone-based surfactant is provided at a v/v concentration range of 0.5-20% of surfactant in oil.

10. The method of claim 1 wherein the bead with the captured amplification copies is sorted for sequencing.

11. The method of claim 1 wherein a plurality of microcapsules when formed each contains on average one or less than one nucleic acid molecule.

12. The method of claim 1 wherein a plurality of microcapsules when formed each contains on average between 5 and 1000 nucleic acid molecules.