Assembly of high fidelity polynucleotides

摘要

Methods and apparatus relate to the synthesis of high fidelity polynucleotides and to the reduction of sequence errors generated during synthesis of nucleic acids on a solid support. Specifically, design of support-bound template oligonucleotides is disclosed. Assembly methods include cycles of annealing, stringent wash and extension of polynucleotides comprising a sequence region complementary to immobilized template oligonucleotides. The error free synthetic nucleic acids generated therefrom can be used for a variety of applications, including synthesis of biofuels and value-added pharmaceutical products.

主权项

1. A method for producing at least one polynucleotide having a predefined sequence, the method comprising the steps of:
(a) providing a support having a plurality of discrete addressable features at predetermined locations of the support, the support comprising at least a first plurality of support-bound oligonucleotides at a first feature and a second plurality of support-bound oligonucleotides at a second feature, wherein the first and second pluralities of support-bound oligonucleotides are single-stranded oligonucleotides and have different predefined sequences, and the first plurality of support-bound oligonucleotides comprises a sequence region at its 5′ end that is the same as a sequence region of a 3′ end of the second plurality of support-bound oligonucleotides; (b) providing a plurality single-stranded oligonucleotides, wherein the 3′ end of the plurality of the single-stranded oligonucleotides is complementary to the 3′ end of the first plurality of support-bound oligonucleotides at selected addressable features; (c) hybridizing the plurality of single-stranded oligonucleotides to the first plurality of support-bound oligonucleotides at the first feature; (d) subjecting the plurality of single-stranded oligonucleotides to a chain extension reaction, thereby forming double-stranded extension products; (e) dissociating the double-stranded extension products, thereby producing a first plurality of single-stranded complementary oligonucleotides; (f) transferring, in a predetermined order, the first plurality of single-stranded complementary oligonucleotides in droplet volume from the first feature at a first predetermined location to the second feature at a second predetermined location of the support, thereby bringing into contact the first plurality of single-stranded complementary oligonucleotides with the second plurality of support-bound oligonucleotides; (g) annealing the first plurality of single-stranded complementary oligonucleotides to the second plurality of support-bound oligonucleotides at the second feature such that the second plurality of support-bound oligonucleotides serves as a template for extension of the first plurality of complementary oligonucleotides in a chain extension reaction, thereby producing the polynucleotide; and (h) optionally amplifying the polynucleotide.