Assembly of high fidelity polynucleotides
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.
1. A method of removing error-containing polynucleotides, the method comprising:
a) providing a plurality of single stranded oligonucleotides, each plurality of oligonucleotides having a predefined sequence, wherein the plurality of oligonucleotides comprise a 5′ end sequence region, a 3′ end sequence region and at least two different sequences regions (N−1) and (N−2) between the 5′ end and the 3′ end sequence regions;
b) providing a plurality of input polynucleotides wherein the plurality of input polynucleotides has at its 3′ end a region that is complementary at least in part to the (N−1) and (N−2) sequences regions of the plurality of oligonucleotides;
c) hybridizing the plurality of input polynucleotides to the plurality of oligonucleotides, thereby generating duplexes;
d) subjecting the duplexes to melt conditions sufficient to denature duplexes having at least one mismatch in a complementary region without denaturing the duplexes that do not comprise a mismatch in the complementary region, thereby releasing a population of error-containing input polynucleotides; and
e) removing error-containing input polynucleotides.
2. The method of claim 1 , wherein each oligonucleotide of the plurality of single-stranded oligonucleotides are bound to a solid support.
3. The method of claim 1 wherein the (N−1) sequence region is adjacent to the 5′ end sequence region and the (N−2) sequence region is adjacent to the (N−1) sequence region.
4. The method of claim 1 wherein the oligonucleotides comprise at least three different sequences regions (N−1), (N−2) and (N−3) between the 5′ end and the 3′ end sequence regions, and wherein the input polynucleotide hybridizes to the (N−1), (N−2) and (N−3) sequences regions of the oligonucleotides.
5. The method of claim 1 wherein the input polynucleotide is a product of at least two consecutive extension chain reactions using the sequences (N−2) and (N−1) as templates.
6. The method of claim 2 wherein each extension cycle is performed at a different feature of the solid support and wherein each extension cycle uses a different plurality of oligonucleotides as template.
7. The method of claim 1 wherein the extension duplexes are subjected to a shuffling process before undergoing a next cycle of extension.
8. The method of claim 7 wherein the shuffling process comprises:
a) denaturing extension duplexes, thereby releasing single-stranded extension products in solution;
b) re-annealing single-stranded extension products to the oligonucleotides, thereby producing re-annealed duplexes;
c) subjecting the re-annealed duplexes to melt conditions sufficient to dissociate error-containing duplexes;
d) removing error-containing single-stranded extension products; and
f) dissociating error-free duplexes, thereby releasing error-free extension products in solution.
9. The method of claim 1 wherein the 3′ end sequence is a spacer sequence.
10. The method of claim 9 wherein the spacer sequence comprises a primer binding site.
11. The method of claim 1 wherein each plurality of oligonucleotides is designed to serve as a template to a different polymerase extension reaction, thereby forming pluralities of extension duplexes, wherein each plurality of extension duplexes has a substantially identical melting temperature.
12. The method of claim 11 wherein the difference of melting temperature between the plurality of duplexes is less than 10° C.
13. The method of claim 11 wherein the difference of melting temperature between the plurality of duplexes is less than 1° C.