Methods and devices for high fidelity polynucleotide synthesis

摘要

Disclosed are methods for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides. 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.

主权项

1. A method for assembling a polynucleotide having a predefined sequence from a plurality of different oligonucleotides, the method comprising:
a) providing a plurality of single-stranded template oligonucleotides at different features on a solid support, wherein each of the plurality of template oligonucleotides comprises a predefined sequence, a primer binding site and a sequence region that is the same or complementary to a sequence region of a single-stranded oligonucleotide present at a different feature of the solid support; b) generating a complementary oligonucleotide for each of the plurality of template oligonucleotides by enzyme-catalyzed synthesis within a primary droplet, thereby producing a plurality of double-stranded oligonucleotides; c) releasing the complementary oligonucleotides from the double-stranded oligonucleotides into the primary droplet; d) combining at least a first and second primary droplets on the support, thereby forming a secondary droplet, wherein the first primary droplet includes a released oligonucleotide that comprises a sequence region that is complementary to a sequence region of a released or template oligonucleotide from the second primary droplet; and e) exposing the secondary droplet to conditions suitable for hybridization and ligation, polymerase extension, or polymerase extension and ligation to assemble a double-stranded polynucleotide having a predefined sequence.