Assays and other reactions involving droplets

Patent metadata

Field	Value
Publication number	US-9816121-B2
Application number	US-201715449637-A
Country	US
Kind code	B2
Filing date	Mar 3, 2017
Priority date	Mar 7, 2007
Publication date	Nov 14, 2017
Grant date	Nov 14, 2017

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.

First claim

What is claimed is:

1. A method for nucleic acid processing, comprising:
(a) directing (i) a first phase comprising reagents, and a plurality of cells, and (ii) a second phase comprising an oil to an intersection of a plurality of channels, wherein said first phase is immiscible with said second phase;
(b) at said intersection, generating a plurality of droplets comprising said plurality of cells and said reagents, wherein an individual droplet of said plurality of droplets comprises a single cell of said plurality of cells, said single cell comprising a first polynucleotide comprising a nucleic acid sequence;
(c) subjecting said first polynucleotide to release from said single cell; and
(d) using said reagents, generating from said first polynucleotide a second polynucleotide comprising a sequence derived from said nucleic acid sequence.

2. The method of claim 1 , further comprising sorting said plurality of droplets to enrich for said individual droplet.

3. The method of claim 1 , wherein said single cell is in a gel capsule.

4. The method of claim 1 , further comprising subjecting said plurality of droplets to conditions sufficient to generate gels in said plurality of droplets, which gels comprise polynucleotides, including said first polynucleotide.

5. The method of claim 1 , wherein (d) comprises performing polymerase chain reaction (PCR) on said first polynucleotide or said second polynucleotide.

6. The method of claim 1 , further comprising sequencing said second polynucleotide or a derivative thereof.

7. A method for nucleic acid processing, comprising:
(a) generating a plurality of droplets each comprising a particle comprising a primer sequence, a single cell comprising a first polynucleotide having a nucleic acid sequence, and reagents;
(b) subjecting said first polynucleotide to release from said single cell; and
(c) using said primer sequence and said reagents, generating from said first polynucleotide a second polynucleotide comprising a sequence derived from said nucleic acid sequence.

8. The method of claim 7 , further comprising subjecting said plurality of droplets to conditions sufficient to generate gels in said plurality of droplets, which gels comprise polynucleotides, including said first polynucleotide.

9. The method of claim 8 , wherein said gels are generated by acrylamide polymerization of said plurality of droplets.

10. The method of claim 7 , wherein said plurality of droplets further comprise cell lysis reagents.

11. The method of claim 7 , wherein each of said plurality of droplets comprises at least 100,000 primer sequences.

12. The method of claim 7 , wherein said reagents comprise reverse transcription reagents.

13. The method of claim 7 , wherein said particle comprises a plurality of copies of said primer sequence.

14. The method of claim 7 , wherein (c) comprises performing polymerase chain reaction (PCR) on said first polynucleotide or said second polynucleotide.

15. The method of claim 7 , further comprising sequencing said second polynucleotide or a derivative thereof.

16. The method of claim 7 , wherein said particle is a bead comprising a plurality of copies of said primer sequence.

17. The method of claim 16 , wherein each of at least a subset of said plurality of droplets comprises a single bead, and wherein said at least said subset of said plurality of droplets are monodisperse.

18. The method of claim 1 , further comprising, in (a), directing said first phase along a first channel of said plurality of channels and said second phase along a second channel of said plurality of channels.

19. The method of claim 18 , further comprising, in (a), directing said plurality of cells along said first channel.

20. The method of claim 1 , wherein said reagents comprise reverse transcription reagents.

21. The method of claim 1 , wherein said reagents comprise a polymerase and dNTPs.

22. The method of claim 7 , wherein said reagents comprise a polymerase and dNTPs.

23. The method of claim 1 , wherein each of at least a subset of said plurality of droplets comprises a single bead, and wherein said at least said subset of said plurality of droplets are monodisperse.

24. The method of claim 7 , wherein said particle is a single particle.

25. The method of claim 1 , wherein said plurality of droplets further comprise cell lysis reagents.

26. The method of claim 7 , further comprising sorting said plurality of droplets.

27. The method of claim 1 , wherein said plurality of droplets is generated without droplet coalescence.

28. The method of claim 7 , wherein said plurality of droplets is generated without droplet coalescence.

29. The method of claim 7 , wherein said single cell is in a gel capsule.

30. The method of claim 1 , wherein said second phase further comprises a surfactant that prevents droplet coalescence.

31. The method of claim 30 , wherein said surfactant comprises a perfluorinated polyether.

32. The method of claim 1 , wherein (d) comprises performing reverse transcription on said first polynucleotide to generate said second polynucleotide.

33. The method of claim 7 , wherein (c) comprises performing reverse transcription on said first polynucleotide to generate said second polynucleotide.

Assignees

• Harvard College

Inventors

• Agresti Jeremy
• Chu Liang-Yin
• Weitz David A
• Kim Jin-Woong
• Rowat Amy
• Sommer Morten
• Dantas Gautam
• Church George

Classifications

• B01L3/502784

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• G01N2015/1006

  for cytology · CPC title

• B01J13/0065

  containing an organic phase · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title