In vitro evolution in microfluidic systems

Patent metadata

Field	Value
Publication number	US-9919277-B2
Application number	US-201715588210-A
Country	US
Kind code	B2
Filing date	May 5, 2017
Priority date	Oct 8, 2004
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired activity; wherein at least one step is under microfluidic control. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

First claim

The invention claimed is:

1. A method for conducting an enzymatic reaction, comprising the steps of:
providing a droplet generator to produce, under microfluidic control, a plurality of aqueous microcapsules surrounded by an immiscible continuous phase that comprises a fluorinated oil that comprises a fluorinated polymer surfactant, each of the plurality of microcapsules comprising an enzyme, a genetic element linked covalently or non-covalently to a bead, and reagents for the enzymatic reaction;
pooling the microcapsules into one or more common compartments such that a portion of the plurality of microcapsules contact each other but do not fuse with each other due to the presence of the surfactant; and
conducting the enzymatic reaction on the genetic element of at least one of the plurality of microcapsules within the one or more common compartments.

2. The method of claim 1 , wherein the genetic element comprises nucleic acids.

3. The method of claim 2 , wherein the nucleic acids further comprise primers for a polymerase chain reaction (PCR).

4. The method of claim 2 , wherein the genetic element comprises RNA.

5. The method of claim 1 , wherein the genetic element is labeled.

6. The method of claim 1 , wherein the microcapsules are monodisperse with respect to each other.

7. The method of claim 1 , wherein the droplet generator further comprises an aqueous fluid channel and two immiscible continuous phase channels and producing the plurality of microcapsules surrounded by the immiscible continuous phase under the microfluidic control comprises partitioning an aqueous fluid that is flowing through the aqueous fluid channel with two counter propagating streams of the immiscible continuous phase that are flowing through the immiscible continuous phase channels.

8. The method of claim 1 , wherein the concentration of the beads is adjusted such that a single bead appears in each microcapsule.

9. The method of claim 1 , wherein the genetic element is identified with a tag.

10. The method of claim 1 , wherein the droplet generator comprises a channel that narrows relative to the direction of flow.

11. The method of claim 1 , wherein the enzymatic reaction is an amplification reaction.

12. The method of claim 11 , wherein the amplification reaction is a strand displacement amplification.

13. The method of claim 1 , wherein the microcapsules are produced by infusing an aqueous fluid through a narrow constriction into a counter-propagating oil stream.

14. The method of claim 1 , wherein the enzymatic reaction is a reverse transcription reaction.

Assignees

• Medical Res Council
• Harvard College

Inventors

• Griffiths Andrew David
• Weitz David A
• Link Darren Roy
• Ahn Keunho
• Bibette Jerome

Classifications

• B01L2300/0867

  Multiple inlets and one sample wells, e.g. mixing, dilution · CPC title

• G01N15/1459

  the analysis being performed on a sample stream · CPC title

• B01J2219/00468

  by manipulation of individual beads · CPC title

• G01N15/1484

  microstructural devices · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title