Microfluidic devices and methods for gene synthesis

• US 9,216,414 B2
• Filed: 11/19/2010
• Issued: 12/22/2015
• Est. Priority Date: 11/25/2009
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Chu, Larry Li-Yang
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US13/511,850
• Publication Number:
  US 20120283140A1
• Time in Patent Office: 1,859 Days
• Field of Search: 435/6.1, 435/91.2, 435/283.1, 435/287.2, 435/288.5, 422/68.1, 536/23.1, 506/16
• US Class Current: 1/1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00655 the compounds being bound to magnets embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/028 Modular arrangements
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• 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: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046

Abstract

Certain aspects of the present invention provide devices and methods for preparing oligonucleotides and for assembling nucleic acid molecules using microfluidic devices.

Claims

1. A microfluidic system comprising:

a continuous carrier phase fluid comprising droplets that are immiscible with the carrier phase fluid;

at least one main channel adapted to carry the carrier phase fluid, the channel comprising interior walls; and

a plurality of defined surface-capture spots forming a pattern on the channel interior walls, at least one of the plurality of defined surface-capture spots comprising oligonucleotides and a surface area modification having a hydrophilic surface property different than the interior walls, wherein the hydrophilic surface property allows droplet absorption and/or attachment, the plurality of defined surface-capture spots comprising oligonucleotides being configured to allow assembly of the oligonucleotides into a target polynucleotide sequence.

2. The microfluidic system of claim 1 further comprising an inlet module upstream of the main channel.

3. The microfluidic system of claim 1 further comprising an outlet module downstream of the main channel.

4. The microfluidic system of claim 1 wherein the surface modification affects the hydrophilic surface property of at least one of the plurality of defined surface-capture spots.

5. The microfluidic system of claim 1 wherein the carrier phase fluid is a water-immiscible fluid, and wherein the channel interior walls comprise an hydrophobic surface.

6. The microfluidic system of claim 1 wherein each of the plurality of defined surface-capture spots comprises oligonucleotides having a different predetermined sequence that together form the target polynucleotide sequence.

7. The microfluidic system of claim 6 wherein the plurality of defined surface-capture spots form a high density oligonucleotide array.

8. The microfluidic system of claim 1 further comprising a surface-capture module, wherein the surface capture module comprises the plurality of defined surface-capture spots and a force transducer.

9. The microfluidic system of claim 8 wherein the force transducer is an electric field, a magnetic field, a mechanical force, an optically induced force or any combination thereof.

10. The microfluidic system of claim 1 further comprising a force transducer for controlling droplet displacement from a first defined surface-capture spot to a second defined surface-capture spot.

11. The microfluidic system of claim 10 wherein the force transducer is an electric field, a magnetic field, a mechanical force, an optically induced force or any combination thereof.

12. The microfluidic system of claim 1 wherein the droplet is water-based and the carrier phase fluid is oil-based.

13. The microfluidic system of claim 1, wherein one or more of the defined surface-capture spots is passivated.

14. The microfluidic system of claim 13, wherein the passivated surface-capture spot is protected with a reagent.

15. The microfluidic system of claim 14, wherein the reagent is oil or short chain hydrocarbon.

16. The microfluidic system of claim 1, further comprising one or more reagents for performing amplification, assembly, and/or error correction, contained within the droplets.

17. A microfluidic system for assembling a target polynucleotide having a predetermined sequence comprising:

a continuous carrier phase fluid comprising droplets that are immiscible with the carrier phase fluid;

a plurality of channels adapted to carry the carrier phase fluid, the channel comprising hydrophobic interior walls;

a plurality of defined surface-capture spots on the interior walls, the surface-capture spots being chemically modified to confer hydrophilic surface property that allows droplet absorption and/or attachment, each defined surface-capture spot comprising oligonucleotides having a different predetermined subsequence that together form the predetermined sequence of the target polynucleotide; and

a force transducer for controlling droplet displacement from a first defined surface-capture spot to a second defined surface-capture spot to allow assembly of the oligonucleotides into the target polynucleotide.

18. The microfluidic system of claim 17 further comprising a reaction chamber for the assembly of the target polynucleotide.

19. The microfluidic system of claim 17 wherein the force transducer is an electric field, a magnetic field, a mechanical force, an optically induced force or any combination thereof.

20. The microfluidic system of claim 17, further comprising one or more one or more reagents for performing amplification, assembly, and/or error correction, contained within the droplets.