Assays and other reactions involving droplets
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.
1. A method, comprising:
(a) providing a plurality of droplets, wherein a droplet of said plurality of droplets comprises a gel particle of a plurality of gel particles, a cell of a plurality of cells, and a nucleic acid molecule of a plurality of nucleic acid molecules, wherein said cell comprises a sample nucleic acid molecule; and
(b) using said nucleic acid molecule and said sample nucleic acid molecule to generate a nucleic acid product.
2. The method of claim 1 , wherein (a) comprises directing a first phase along a channel of a microfluidic device to contact a second phase to generate said plurality of droplets.
3. The method of claim 2 , wherein said first phase comprises said plurality of cells.
4. The method of claim 1 , wherein said gel particle is configured to liquefy upon application of a stimulus.
5. The method of claim 4 , wherein said stimulus comprises one or more members selected from the group consisting of a change in temperature, pH, and osmotic pressure.
6. The method of claim 1 , wherein said plurality of droplets is monodisperse.
7. The method of claim 1 , wherein said plurality of gel particles is monodisperse.
8. The method of claim 1 , wherein said plurality of gel particles comprise polyacrylamide.
9. The method of claim 1 , wherein said droplet further comprises nucleic acid amplification reagents.
10. The method of claim 9 , wherein said nucleic acid amplification reagents comprise a polymerase and nucleotides.
11. The method of claim 10 , wherein (b) further comprises using said nucleic acid amplification reagents to generate said nucleic acid product.
12. The method of claim 11 , wherein said polymerase is a Phi29 polymerase.
13. The method of claim 1 , further comprising providing a surfactant that prevents droplet coalescence, wherein said surfactant is selected from the group consisting of Span80, Span80/Tween-20, Span80/Triton X-100, Abil EM90, Abil we09, polyglycerol polyricinoleate, Tween-85, 749 Fluid, and an ammonium carboxylate salt of Krytox.
14. The method of claim 1 , wherein said plurality of gel particles comprises said plurality of nucleic acid molecules, and wherein said gel particle comprises said nucleic acid molecule.
15. The method of claim 1 , wherein said sample nucleic acid molecule comprises a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule.
16. The method of claim 1 , wherein said sample nucleic acid molecule comprises a RNA molecule, and wherein said nucleic acid product in (b) is generated by performing reverse transcription PCR using said RNA molecule.
17. The method of claim 1 , wherein said sample nucleic acid molecule comprises a DNA molecule, and wherein said nucleic acid product in (b) is generated by performing a PCR reaction using said DNA molecule.
18. The method of claim 1 , wherein said nucleic acid product in (b) is generated by performing whole-genome amplification using said sample nucleic acid molecule.
19. The method of claim 1 , wherein said plurality of nucleic acid molecules comprises at least 10 5 species.
20. The method of claim 1 , wherein said plurality of nucleic acid molecules comprises at least 10 6 species.
21. The method of claim 1 , wherein said plurality of nucleic acid molecules comprises a DNA library.
22. The method of claim 1 , wherein said plurality of nucleic acid molecules comprises a RNA library.
23. The method of claim 1 , wherein said plurality of droplets comprises a protein library.
24. The method of claim 1 , wherein said cell comprises a whole genome and wherein said sample nucleic acid molecule comprises a region of said whole genome.
25. The method of claim 24 , wherein said region of said whole genome comprises a polymorphism.
26. The method of claim 1 , further comprising thermally cycling said droplet.
27. The method of claim 1 , wherein (b) comprises causing said gel particle to liquefy, thereby allowing said nucleic acid molecule to interact with said sample nucleic acid molecule.
28. The method of claim 27 , wherein causing said gel particle to liquefy comprises exposing said gel particle to an environmental change.
29. The method of claim 28 , wherein said environmental change comprises one or more members selected from the group consisting of a change in temperature, a change in pH, and a change in osmotic pressure.
30. The method of claim 1 , wherein said plurality of droplets is monodisperse.