Methods and devices for nucleic acids synthesis
Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.
1. A method for synthesizing a plurality of polynucleotides having a predefined sequence, the method comprising:
a. providing a first support comprising a plurality of features, wherein each feature comprises a plurality of support-bound anchor single-stranded oligonucleotides, wherein the 5′ end of each of the plurality of the anchor oligonucleotides is complementary to the 5′ end of a first plurality of construction oligonucleotides;
b. providing a second support having a plurality of features, wherein each feature comprises a plurality of single-stranded support-bound template oligonucleotides, wherein the plurality of single-stranded support-bound template oligonucleotides comprises at least two populations of oligonucleotides having different predefined sequences, the at least two populations of oligonucleotides having 3′ end complementary sequences,
c. generating at least a first and second plurality of construction oligonucleotides by polymerase extension using the plurality of single-stranded support-bound template oligonucleotides as templates, wherein the first plurality of construction oligonucleotides are complementary to the first population of oligonucleotides, and wherein the second plurality of construction oligonucleotides are complementary to the second population of oligonucleotides;
d. positioning the first and the second support such that each feature of the second support is aligned to a corresponding feature of the first support;
e. releasing the at least first and second pluralities of construction oligonucleotides in a solution under conditions promoting hybridization of the first plurality of construction oligonucleotides to the plurality of corresponding anchor oligonucleotides and hybridization of the second plurality of construction oligonucleotides to the first plurality of construction oligonucleotides.
2. The method of claim 1 , wherein the step of generating at least the first and second plurality of construction oligonucleotides comprises annealing a primer sequence having at least one uracil to the plurality of single-stranded support-bound template oligonucleotides under conditions promoting extension of the primer and removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.
3. The method of claim 1 , comprising further ligating the anchor oligonucleotides and pluralities of construction oligonucleotides.
4. The method of claim 1 , wherein the second support is positioned above and facing the first support.
5. The method of claim 4 , wherein step of releasing the at least first and second plurality of construction oligonucleotides in solution allows for the diffusion of the at least first and second plurality of construction oligonucleotides towards the anchor oligonucleotides.
6. The method of claim 5 , wherein the step of releasing the at least first and second plurality of construction oligonucleotides in solution is in the presence of a porous membrane allowing for a substantial vertical diffusion of the construction oligonucleotides towards the anchor oligonucleotides.
7. The method of claim 6 , wherein the porous membrane decreases the lateral diffusion of construction oligonucleotides.
8. The method of claim 1 , wherein the solution comprises a ligase.
9. The method of claim 1 , wherein the stoichiometry of each of the at least first and second plurality of construction oligonucleotides is higher than the stoichiometry of the anchor oligonucleotides.
10. The method of claim 1 , further comprising exposing the plurality of polynucleotides to a mismatch recognizing and cleaving component under conditions suitable for cleavage of double-stranded polynucleotides containing a mismatch nucleotide.
11. The method of claim 10 , wherein the plurality of polynucleotides is immobilized on the support or is in solution.
12. The method of claim 11 , wherein the mismatch recognizing and cleaving component comprises a mismatch endonuclease.
13. The method of claim 12 , wherein the mismatch specific endonuclease is a CEL I enzyme.