Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly

Patent metadata

Field	Value
Publication number	US-10207240-B2
Application number	US-201013505646-A
Country	US
Kind code	B2
Filing date	Nov 3, 2010
Priority date	Nov 3, 2009
Publication date	Feb 19, 2019
Grant date	Feb 19, 2019

Abstract

Methods and devices are provided for manipulating droplets on a support using surface tension properties, moving the droplets along a predetermined path and merging two droplets together enabling a number of chemical reactions. Disclosed are methods for controlling the droplets volumes. Disclosed are methods and devices for synthesizing at least one oligonucleotide having a predefined sequence. Disclosed are methods and devices for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides having a predefined sequence. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonucleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.

First claim

What is claimed is:

1. A composition for nucleic acid synthesis, the composition comprising:
water-in-oil emulsion sub-microvolume droplets for synthesis of a target nucleic acid comprising an aqueous phase, an immiscible oil phase, and a surfactant and/or other stabilizing molecules to maintain the integrity of the droplets,
wherein the synthesis of target nucleic acid occurs within the aqueous phase of the water-in-oil emulsion sub-microvolume droplet,
wherein the aqueous phase of each water-in-oil emulsion sub-microvolume droplet comprises a plurality of single-stranded support-bound oligonucleotides each having a predefined sequence, a plurality of single-stranded oligonucleotides in solution having complementary sequences to a terminal region of the plurality of single stranded support-bound oligonucleotides, a thermostable polymerase, a thermostable ligase and a glycerol, wherein glycerol increases the boiling point of the aqueous phase of the water-in-oil emulsion sub-microvolume droplets;
wherein each water-in-oil emulsion sub-microvolume droplet has an aqueous volume of 0.5 picoliters to 100 nanoliters, and
wherein the plurality of single-stranded oligonucleotides in solution together comprise the target polynucleotide.

2. The composition of claim 1 wherein the plurality of single-stranded support-bound oligonucleotides are bound to a solid support selected from the group consisting of a pin, a strip, a plate, a disk, an array, a rod, a particle and a nanoparticle.

3. The composition of claim 1 wherein the plurality of single-stranded support-bound oligonucleotides are bound to one or more beads.

4. The composition of claim 1 wherein the plurality of single-stranded support-bound oligonucleotides have different predefined sequences.

5. The composition of claim 1 wherein the aqueous phase of water-in-oil emulsion sub-microvolume droplet further comprises one or more of deoxyadenosine 5′-triphosphate, deoxythymidine 5′-triphosphate, deoxycytidine 5′-triphosphate, and deoxyguanosine 5′-triphosphate.

6. The composition of claim 1 wherein the aqueous phase of water-in-oil emulsion sub-microvolume droplet further comprises at least one primer.

7. A composition for synthesis of a target polynucleotide comprising:
one or more water-in-oil emulsion sub-microvolume droplets comprising an aqueous phase, an immiscible oil phase, and a surfactant and/or other stabilizing molecules to maintain the integrity of the droplets, wherein the synthesis of the target polynucleotide occurs within the aqueous phase of the one or more water-in-oil emulsion sub-mircovolume droplets;
a plurality of support-bound oligonucleotides contained in the aqueous phase of one or more water-in-oil emulsion sub-microvolume droplets, each support-bound oligonucleotide having a predefined sequence and having a terminal region comprising a sequence identical to a terminal region of another oligonucleotide, the plurality of oligonucleotides together comprise the target polynucleotide;
wherein the aqueous phase of each of the one or more water-in-oil emulsion sub-microvolume droplets further comprises a thermostable polymerase, a primer, deoxyribonucleoside triphosphates, a thermostable ligase and glycerol, wherein glycerol increases the boiling point of the water-in-oil emulsion sub-microvolume droplets and
wherein each water-in-oil emulsion sub-microvolume droplet has an aqueous volume of 0.5 picoliters to 100 nanoliters.

8. The composition of claim 7 wherein the plurality of single-stranded support-bound oligonucleotides are bound to a solid support selected from the group consisting of a pin, a strip, a plate, a disk, an array, a rod, a particle and a nanoparticle.

9. The composition of claim 7 wherein the plurality of support-bound oligonucleotides are single-stranded oligonucleotides having different predefined sequences.

10. The composition of claim 7 wherein the primer is a unique primer or a universal primer.

11. A composition for synthesis of a target polynucleotide comprising:
one or more water-in-oil emulsion sub-microvolume droplets comprising an aqueous phase, an immiscible oil phase, and a surfactant and/or other stabilizing molecules to maintain the integrity of the droplets, wherein the synthesis of the target polynucleotide occurs within the aqueous phase of the one or more water-in-oil emulsion sub-microvolume droplets;
one or more pluralities of single-stranded support-bound oligonucleotides and one or more pluralities of single-stranded oligonucleotides in solution in the aqueous phase of the one or more water-in-oil emulsion sub-microvolume droplets, each plurality of single-stranded oligonucleotides having a different predefined sequence;
wherein the aqueous phase of the one or more water-in-oil emulsion sub-microvolume droplets further comprises a thermostable polymerase, a primer, deoxyribonucleosides triphosphates, a thermostable ligase, and glycerol, wherein glycerol increases the boiling point of the aqueous phase of the water-in-oil emulsion sub-microvolume droplets,
wherein each of the one or more water-in-oil emulsion sub-microvolume droplets has an aqueous volume of 0.5 picoliters to 100 nanoliters, and
wherein a first single-stranded oligonucleotide in solution has a terminal region comprising a sequence complementary to a sequence of a terminal region of the support-bound oligonucleotide, and wherein the first single-stranded oligonucleotide in solution has a terminal region comprising a sequence complementary to a sequence of a terminal region of a second single-stranded oligonucleotide in solution, the plurality of single-stranded oligonucleotides in solution together comprising the target polynucleotide.

12. The composition of claim 11 wherein each of the one or more plurality of single-stranded support-bound oligonucleotides are bound to a solid support selected from the group consisting of a pin, a strip, a plate, a disk, an array, a rod, a particle and a nanoparticle.

Assignees

• Jacobson Joseph
• Chu Larry Li Yang
• Ramu Senthil
• Gen9 Inc

Inventors

• Jacobson Joseph
• Chu Larry Li-Yang
• Ramu Senthil

Classifications

• B01J2219/00608

  DNA chips · CPC title

• B01J2219/00659

  Two-dimensional arrays · CPC title

• B01L2400/0454

  radiation pressure, optical tweezers · CPC title

• B01L3/5088

  confining liquids at a location by surface tension, e.g. virtual wells on plates, wires (B01L3/50857 takes precedence) · CPC title

• B01J2219/00378

  Piezoelectric or ink jet dispensers · CPC title