Methods and devices for high fidelity polynucleotide synthesis
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.
1. A method for synthesizing a plurality of oligonucleotides having a predefined sequence, the method comprising:
(a) providing a plurality of template oligonucleotides in a solution comprising a primer, a polymerase and nucleotides, wherein each of the plurality of template oligonucleotides is bound on a solid support comprising a predefined subsequence and a primer binding site for the primer, and wherein the primer comprises at least one nuclease recognition site for a nuclease, wherein all predefined subsequences are designed such that together they form a target having a predefined sequence;
(b) exposing the plurality of template oligonucleotides to conditions suitable for primer hybridization and polymerase extension, thereby producing a plurality of complementary oligonucleotides;
(c) releasing the plurality of complementary oligonucleotides;
(d) exposing the plurality of complementary oligonucleotides to the nuclease under conditions suitable for the nuclease to bind to the nuclease recognition site and cleave the primer from the plurality of complementary oligonucleotides;
(e) exposing the cleaved complementary oligonucleotides and the template oligonucleotides to conditions suitable for hybridization, thereby producing a plurality of partially double-stranded oligonucleotides;
(f) washing the plurality of partially double-stranded oligonucleotides to remove the cleaved primers;
(g) releasing the cleaved complementary oligonucleotides into at least two droplets; and
(h) merging the at least two droplets thereby bringing the plurality of cleaved complementary oligonucleotides together to assemble the target having the predefined sequence.
2. The method of claim 1 , wherein the at least one nuclease recognition site permits cleavage between the primer and the complementary oligonucleotide.
3. The method of claim 1 , wherein the primer comprises at least two nuclease recognition sites.
4. The method of claim 1 , wherein the primer comprises a first and at least a second nuclease recognition site, wherein the first recognition site permits cleavage between the primer and the complementary oligonucleotide and the at least second nuclease recognition site is located within the primer.
5. The method of claim 4 , further comprising exposing the primer to the at least second nuclease under conditions suitable for the second nuclease to bind to the primer and subject the primer to cleavage.
6. The method of claim 1 , wherein the primers comprise Uracil and the nuclease is a Uracil DNA glycosylase.
7. The method of claim 1 , wherein the plurality of template oligonucleotides includes a universal primer binding site.
8. The method of claim 1 , wherein at least two template oligonucleotides have different primer binding sites.
9. The method of claim 1 , wherein each of the plurality of template oligonucleotides is provided on a different feature of a support.
10. The method of claim 9 , wherein the plurality of complementary oligonucleotides is produced for each of the plurality of template oligonucleotides at the different feature of the support.