Partition processing methods and systems

• US 10,697,000 B2
• Filed: 02/23/2016
• Issued: 06/30/2020
• Est. Priority Date: 02/24/2015
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Belgrader, Phillip, Masquelier, Donald A
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/050,948
• Publication Number:
  US 20160244809A1
• Time in Patent Office: 1,589 Days
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 2001/4038 electric methods, e.g. electromigration, electrophoresis, ionisation

Abstract

The disclosure provides methods for separating and/or purifying one or more molecules released from one or more fluid compartments or partitions, such as one or more droplets. Molecules can be released from a fluid compartment(s) and bound to supports that can be isolated via any suitable method, including example methods described herein. The disclosure also provides devices that can aid in isolating supports bound to molecules.

Claims

1. A method for purifying a target molecule, comprising:

a) providing a magnetic separation device comprising a first magnetic source and a second magnetic source, wherein said magnetic separation device comprises;

i) a first receptacle disposed on a first face of said magnetic separation device, wherein said first receptacle is configured to position a vessel relative to a first magnetic source; and

ii) a second receptacle disposed on a second face of said magnetic separation device, wherein said second receptacle is configured to position said vessel relative to a second magnetic source, wherein said second face of said magnetic separation device is different from said first face of said magnetic separation device, and wherein said second receptacle of said magnetic separation device is different from said first receptacle of said magnetic separation device;

b) in said vessel, providing a support to a liquid mixture comprising contents of a destabilized droplet that comprise a target molecule, wherein said support binds said target molecule to provide a bound target molecule;

c) positioning said vessel in said first receptacle, such that said first magnetic source provides a magnetic field that immobilizes said support at a first location within said vessel, wherein said magnetic separation device is at a first orientation;

d) while said support is immobilized at said first location, removing said liquid mixture from said vessel;

e) providing a suspension fluid to said vessel, thereby suspending said support in said suspension fluid;

f) moving said magnetic separation device from said first orientation to a second orientation different from said first orientation; and

g) positioning said vessel in said second receptacle such that said second magnetic source provides a magnetic field that immobilizes said support at a second location within said vessel, wherein said magnetic separation device is at said second orientation,

wherein said second location within said vessel is different than said first location within said vessel.

2. The method of claim 1, wherein, in b), said liquid mixture further comprises a chaotrope that aids in binding of said support to said target molecule.

3. The method of claim 2, wherein said chaotrope comprises guanidine thiocyanate or guanidine hydrochloride.

4. The method of claim 1, wherein, in b), said liquid mixture further comprises a polymeric species.

5. The method of claim 4, wherein said polymeric species comprises polyacrylamide or agarose.

6. The method of claim 5, wherein said polyacrylamide comprises a linear polyacrylamide.

7. The method of claim 1, wherein, in b), said destabilized droplet is an aqueous droplet.

8. The method of claim 1, wherein, in b), said liquid mixture further comprises a destabilization agent capable of destabilizing an emulsion.

9. The method of claim 8, wherein said destabilization agent comprises perfluorooctanol.

10. The method of claim 1, wherein, in b), said liquid mixture further comprises a primer.

11. The method of claim 10, wherein said primer comprises a barcode sequence.

12. The method of claim 10, wherein said primer comprises a random N-mer.

13. The method of claim 1, wherein, in b), said liquid mixture further comprises a reducing agent.

14. The method of claim 1, wherein d) further comprises removing said liquid mixture from said vessel via suction.

15. The method of claim 1, wherein d) further comprises removing said liquid mixture from said vessel via decanting.

16. The method of claim 1, wherein, in e), said suspension fluid comprises ethanol, isopropanol, or acetone.

17. The method of claim 1, further comprising, after g), releasing said bound target molecule from said support, to provide a released target molecule.

18. The method of claim 17, wherein said releasing is completed with the aid of an elution agent.

19. The method of claim 18, wherein said elution agent is selected from the group consisting of water, Tris buffer, phosphate buffer, and sodium hydroxide.

20. The method of claim 17, prior to said releasing said bound target molecule from said support, washing said support in one or more wash cycles by contacting said support with a washing agent.

21. The method of claim 20, wherein said washing agent comprises ethanol, isopropanol, or acetone.

22. The method of claim 17, further comprising, after said releasing said bound target molecule from said support, subjecting said released target molecule to a solid phase reversible immobilization process.

23. The method of claim 1, wherein said target molecule comprises a target nucleic acid molecule.

24. The method of claim 23, further comprising, after g), determining a sequence of said target nucleic acid molecule.

25. The method of claim 23, further comprising, after g), appending one or more additional nucleotides to said target nucleic acid molecule to provide a larger target nucleic acid molecule.

26. The method of claim 25, further comprising determining a sequence of said larger target nucleic acid molecule.

27. The method of claim 1, wherein said target molecule comprises a small molecule, a protein, or a peptide.

28. The method of claim 1, wherein said support comprises a magnetic material.

29. The method of claim 1, wherein said support comprises a particle.

30. The method of claim 1, wherein said support is functionalized with a silanol that aids said support in binding said target molecule.

31. The method of claim 1, wherein said support is functionalized with a carboxylate that aids said support in binding said target molecule.

32. The method of claim 1, wherein said vessel is selected from the group consisting of a tube, a well, a dish and a container.

33. The method of claim 1, wherein g) further comprises repositioning said magnetic separation device prior to positioning said vessel in said second receptacle of said magnetic separation device.

34. The method of claim 33, wherein repositioning said magnetic separation device comprises rotating said device or flipping said device over.

35. The method of claim 1, wherein said first magnetic source and said second magnetic source are the same magnetic source.