ASSAYS AND OTHER REACTIONS INVOLVING DROPLETS

• US 20170183701A1
• Filed: 03/03/2017
• Published: 06/29/2017
• 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: Church, George, Weitz, David A., Rowat, Amy, Sommer, Morten, Kim, Jin-Woong, Agresti, Jeremy, Chu, Liang-Yin, Dantas, Gautam
• Granted Patent: US 9,816,121 B2
• Time in Patent Office: Days
• 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 for nucleic acid processing, comprising:

a. directing a first phase comprising amplification reagents along a first channel and a second phase comprising an oil along a second channel to an intersection of said first channel and said second channel, wherein said first phase is immiscible with said second phase;

b. at said intersection, generating a plurality of droplets comprising individual droplets each having (i) a single cell comprising a first polynucleotide comprising a nucleic acid sequence, and (ii) said amplification reagents; and

c. using said amplification reagents, subjecting said first polynucleotide to nucleic acid amplification to generate a second polynucleotide comprising a sequence derived from said nucleic acid sequence.

2. The method of claim 1, wherein said plurality of droplets further comprises an identifier.

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

4. The method of claim 1, further comprising releasing said first polynucleotide from said single cell prior to (c).

5. The method of claim 1, further comprising hardening said cell into a gel capsule surrounding said cell.

6. 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.

7. The method of claim 1, wherein said nucleic acid amplification is polymerase chain reaction (PCR).

8. The method of claim 1, further comprising generating a sequence of said second polynucleotide.

9. 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 nucleic acid amplification reagents;

b. releasing said first polynucleotide from said cell; and

c. using said primer sequence and said reagents, subjecting said first polynucleotide to nucleic acid amplification to generate a second polynucleotide comprising a sequence derived from said nucleic acid sequence.

10. The method of claim 9, 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.

11. The method of claim 10, wherein said gels are generated by acrylamide polymerization.

12. The method of claim 9, wherein said plurality of droplets further comprise cell lysis reagents.

13. The method of claim 9, wherein each of said plurality of droplets comprises at least 100,000 primer sequences.

14. The method of claim 9, wherein said nucleic acid amplification reagents comprise reverse transcriptase amplification reagents.

15. The method of claim 9, wherein said particles comprises a plurality of copies of said primer sequence.

16. The method of claim 9, wherein said nucleic acid amplification is polymerase chain reaction (PCR).

17. The method of claim 9, further comprising generating a sequence of said second polynucleotide.

18. The method of claim 9, wherein said particle is a bead comprising a plurality of copies of said primer sequence.

19. The method of claim 18, wherein at least a subset of said plurality of droplets comprise a single bead, and wherein said at least said subset of said plurality of droplets are different.

20. The method of claim 19, wherein said at least said subset of said plurality of droplets comprise at least 10⁵ different beads.