Assays and other reactions involving droplets

• US 9,017,948 B2
• Filed: 02/04/2014
• Issued: 04/28/2015
• Est. Priority Date: 03/07/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Agresti, Jeremy, Chu, Liang-Yin, Weitz, David A., Kim, Jin-Woong, Rowat, Amy, Sommer, Morten, Dantas, Gautam, Church, George
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/172,266
• Publication Number:
  US 20140199730A1
• Time in Patent Office: 448 Days
• US Class Current: 435/6.12
• CPC Class Codes: B01F 2101/23 Mixing of laboratory samples e.g. in preparation of analysing or testing properties of materials
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 23/4105 Methods of emulsifying
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01J 13/0052 Preparation of gels
• CPC Class Codes: B01J 13/0065 containing an organic phase
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 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 Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 2015/1006 for cytology

Abstract

The present invention generally relates to droplets and/or emulsions, such as multiple emulsions. In some cases, the droplets and/or emulsions may be used in assays, and in certain embodiments, the droplet or emulsion may be hardened to form a gel. In some aspects, a heterogeneous assay can be performed using a gel. For example, a droplet may be hardened to form a gel, where the droplet contains a cell, DNA, or other suitable species. The gel may be exposed to a reactant, and the reactant may interact with the gel and/or with the cell, DNA, etc., in some fashion. For example, the reactant may diffuse through the gel, or the hardened particle may liquefy to form a liquid state, allowing the reactant to interact with the cell. As a specific example, DNA contained within a gel particle may be subjected to PCR (polymerase chain reaction) amplification, e.g., by using PCR primers able to bind to the gel as it forms. As the DNA is amplified using PCR, some of the DNA will be bound to the gel via the PCR primer. After the PCR reaction, unbound DNA may be removed from the gel, e.g., via diffusion or washing. Thus, a gel particle having bound DNA may be formed in one embodiment of the invention.

Claims

1. A method of amplification comprising:

a. generating a first plurality of droplets comprising polynucleotide primers, wherein the first plurality of droplets excludes polynucleotides to be amplified;

b. generating a second plurality of droplets comprising polynucleotides to be amplified, wherein the polynucleotides are obtained from a whole genome;

c. merging first droplets of the first plurality of droplets with second droplets of the second plurality of droplets to form coalesced droplets; and

d. performing amplification reactions on the polynucleotides in the coalesced droplets to form amplicons.

2. The method of claim 1, wherein the amplification reactions comprise amplifying a plurality of different regions of the polynucleotides obtained from the whole genome.

3. The method of claim 1, wherein the amplification reaction is performed using Phi29 polymerase.

4. The method of claim 1, wherein the whole genome comprises a polymorphism.

5. The method of claim 1, wherein the whole genome is a mammalian genome.

6. The method of claim 1, wherein the whole genome is a primate genome.

7. The method of claim 1, wherein the polynucleotides obtained from the whole genome are DNA.

8. The method of claim 1, wherein the polynucleotides obtained from the whole genome are RNA.

9. The method of claim 1, wherein the first plurality of droplets further comprise amplification reagents.

10. The method of claim 1, wherein the second plurality of droplets further comprise amplification reagents.

11. The method of claim 1, further comprising sequencing the amplicons.

12. The method of claim 1, wherein the amplification reactions are selected from the group consisting of:

Polymerase Chain Reaction (PCR), reverse-transcription PCR and real-time PCR.

13. The method of claim 12, wherein the amplification reactions are reverse transcription PCR.

14. The method of claim 1, wherein the polynucleotide primers comprise an acrydite moiety.

15. The method of claim 1, further comprising hardening the coalesced droplets to form gels.

16. The method of claim 15, wherein the polynucleotide primers are bound to the gels.

17. The method of claim 15, wherein the hardening comprises introducing a gelation initiator.

18. The method of claim 17, wherein the gelation initiator co-flows with an aqueous phase.

19. The method of claim 17, wherein the gelation initiator co-flows with an oil phase.

20. The method of claim 17, wherein the gelation initiator is selected from the group consisting of ammonium persulfate, TEMED and Ca²⁺.

21. The method of claim 15, wherein the gels comprise poly(acrylamide).

22. The method of claim 1, wherein the method has a rate of coalescence of at least 10 droplets per second.

23. The method of claim 1, wherein the average diameter of the first, second or coalesced droplets is at least 1 micrometer.

24. The method of claim 1, wherein the first plurality of droplets comprises a plurality of identical polynucleotide primers.

25. The method of claim 1, wherein some of the droplets in the first plurality of droplets are distinct.

26. The method of claim 1, wherein the amplification reactions are performed using primers from the first droplets.

27. The method of claim 1, wherein the merging of the first droplets with the second droplets is controlled.

28. The method of claim 27, wherein the merging of the first droplets with the second droplets is electrically controlled.

29. The method of claim 17, wherein the gelation initiator comprises a reducing agent.

30. The method of claim 1, wherein the first, second or coalesced droplets comprise an identifier.

31. The method of claim 1, wherein droplets within the first plurality of droplets comprise different identifiers.

32. The method of claim 31, wherein the identifier is an optical identifier.

33. The method of claim 31, wherein the identifier is a fluorophore.

34. The method of claim 1, wherein the first plurality of droplets or the second plurality of droplets comprise aqueous droplets within an oil phase.

35. The method of claim 34, wherein the oil phase comprises fluorocarbon oil.

36. The method of claim 35, wherein the fluorocarbon oil comprises Krytox.

37. The method of claim 1, wherein the first, second or coalesced droplets comprise a fluoro surfactant.

38. The method of claim 1, wherein the first, second or coalesced droplets comprise an ammonium carboxylate salt surfactant.

39. The method of claim 1, wherein the first, second or coalesced droplets comprise a surfactant selected from the group consisting of Span80, Span80/Tween-20, Span80/Triton X-100, Abil EM90, Abil we09, polyglycerol polyricinoleate “

PGPR90”

, Tween-85, 749 Fluid, and ammonium carboxylate salt of Krytox.