System and method for emulsion breaking and recovery of biological elements

• US 8,609,339 B2
• Filed: 09/10/2010
• Issued: 12/17/2013
• Est. Priority Date: 10/09/2009
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Suo, Yue
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US12/879,493
• Publication Number:
  US 20110087016A1
• Time in Patent Office: 1,194 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

An embodiment of a method for extracting biological material from an emulsion is described that comprises the steps of a) breaking an emulsion comprising a plurality of aqueous droplets in a continuous phase of an oil using a solvent to produce a combined aqueous-oil mixture, where the solvent disrupts the aqueous droplets which release a plurality of biological elements each immobilized on a substrate into the combined aqueous-oil mixture; b) introducing an inorganic salt to the combined aqueous-oil mixture causing a phase separation of the mixture into a first phase comprising an aqueous solution and the biological elements and a second phase comprising the solvent and the oil; c) extracting the first phase from the second phase; and d) collecting the substrate immobilized biological elements from the first phase.

Claims

1. A method for extraction of solid-phase immobilized nucleic acids from an emulsion, comprising the steps of:

a) breaking an emulsion comprising a plurality of aqueous droplets in a continuous phase of an oil by application of an organic solvent to produce a combined aqueous-oil mixture, wherein the organic solvent disrupts the aqueous droplets releasing a plurality of nucleic acids each immobilized on a bead substrate into the broken emulsion mixture;

b) introducing an inorganic salt to the broken emulsion mixture causing a phase separation of the mixture into a first phase comprising an aqueous solution and the bead substrate immobilized nucleic acids and a second phase comprising the solvent and the oil;

c) extracting the first phase from the second phase; and

d) collecting the bead substrate immobilized nucleic acids from the first phase.

2. The method of claim 1, wherein:

the organic solvent is selected from the group consisting of isopropanol and ethanol.

3. The method of claim 1, wherein:

the bead substrate is hydrophilic.

4. The method of claim 1, further comprising:

a plurality of the bead substrates each comprising a population of nucleic acids obtained by a previously executed process of nucleic acid amplification from a single nucleic acid molecule within one of the aqueous droplets, wherein the population of nucleic acids is attached during the process of amplification onto the surface of the bead.

5. The method of claim 1, wherein:

the inorganic salt is selected from the group consisting of NaCl, KCl, LiCl, Na₂SO₄, potassium carbonate, and ammonium sulfate.

6. The method of claim 1, wherein:

the inorganic salt is introduced in a granular form.

7. The method of claim 1, wherein:

the inorganic salt is introduced in a solution form.

8. The method of claim 1, further comprising wherein:

the bead substrate immobilized nucleic acids are collected in step d) by filtration of the first phase obtained in step c).

9. The method of claim 1, further comprising:

introducing a liquid material which is capable of forming a third phase between the first phase and the second phase to produce a gap phase.

10. The method of claim 9, wherein:

the liquid material comprises a silicone oil.