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