ASSAYS AND OTHER REACTIONS INVOLVING DROPLETS

• US 20140199730A1
• Filed: 02/04/2014
• Published: 07/17/2014
• 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
• Granted Patent: US 9,017,948 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• 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 whole genome amplification comprising:

a. generating a first plurality of droplets comprising polynucleotide primers;

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

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

d. performing a whole genome amplification reaction on the polynucleotides in the coalesced droplet to form amplicons.

2. The method of claim 1, wherein the amplification reaction comprises amplifying a plurality of different regions of the polynucleotides obtained from the 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 reaction is 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 reaction is 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 droplet to form a gel.

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

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 gel is 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 droplet is at least 1 micrometer.