Microfluidic devices and methods for gene synthesis

• US 9,968,902 B2
• Filed: 11/20/2015
• Issued: 05/15/2018
• Est. Priority Date: 11/25/2009
• Status: Expired due to Fees

• 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:
  US14/946,811
• Publication Number:
  US 20160144332A1
• Time in Patent Office: 907 Days
• 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 at least one droplet in fluid communication with a solid support, wherein the at least one droplet is immiscible with the carrier phase fluid and wherein the at least one droplet comprises at least one input oligonucleotide, wherein the input oligonucleotide has sequence complementarity or overlaps with at least one immobilized oligonucleotide;

a plurality of defined surface-capture spots forming a pattern on the solid support,

wherein at least one of the plurality of defined surface-capture spots comprises a plurality of immobilized oligonucleotides and a patch having a hydrophilic surface property different than the solid support wherein the hydrophilic surface property allows droplet absorption and/or attachment; and

wherein the plurality of defined surface-capture spots comprising immobilized oligonucleotides being configured to allow assembly of the input oligonucleotides into a target polynucleotide sequence.

2. The microfluidic system of claim 1, further comprising at least one main channel adapted to carry the carrier phase fluid.

3. The microfluidic system of claim 2, wherein the channel comprises an interior wall.

4. The microfluidic system of claim 3, wherein the plurality of defined surface-capture spots form a pattern on the interior wall.

5. The microfluidic system of claim 3, wherein at least one defined surface-capture spot comprises a patch having a hydrophilic surface property different than the interior wall, wherein the hydrophilic surface property allows droplet absorption and/or attachment.

6. The microfluidic system of claim 2, further comprising an inlet module upstream of the main channel and/or an outlet module downstream of the main channel.

7. The microfluidic system of claim 1, wherein the carrier phase fluid is water immiscible.

8. The microfluidic system of claim 7, wherein the solid support comprises a hydrophobic surface and wherein the defined surface-capture spots are hydrophilic.

9. The microfluidic system of claim 1, wherein the solid support is a high density oligonucleotide array.

10. The microfluidic system of claim 1, further comprising a force transducer that controls 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 at least one droplet is water-based and the carrier phase fluid is oil-based.

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

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

a continuous carrier phase fluid comprising at least one droplet in fluid communication with a solid support, wherein the at least one droplet is immiscible with the carrier phase fluid and comprises at least one input oligonucleotide;

a plurality of defined surface-capture spots forming a pattern wherein the solid support comprises immobilized oligonucleotides that together comprise a target polynucleotide and wherein the input oligonucleotide has sequence complementarity or overlaps with at least one immobilized oligonucleotide; and

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

15. The microfluidic system of claim 14, wherein at least one defined surface-capture spots forming a pattern comprises a patch having a hydrophilic surface property different than the solid support, wherein the hydrophilic surface property allows droplet absorption and/or attachment.

16. The microfluidic system of claim 14, wherein the solid support is a high density oligonucleotide array.

17. The microfluidic system of claim 14, wherein the force transducer is an electric field, a magnetic field, a mechanical force, an optically induced force or any combination thereof.

18. The microfluidic system of claim 14, further comprising one or more one or more reagents for performing amplification, assembly, and/or error correction, contained within the droplets.