Methods and Apparatuses for Chip-Based DNA Error Reduction

• US 20120028843A1
• Filed: 06/20/2011
• Published: 02/02/2012
• Est. Priority Date: 11/25/2009
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Ramu, Senthil
• Granted Patent: US 9,422,600 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Methods and apparatus relate to reduction of sequence errors generated during synthesis of nucleic acids on a microarray chip. The error reduction can include synthesis of complementary stands (to template strands), using a short universal primer complementary to the template strands and polymerase. Heteroduplex can be formed be melting and re-annealing complementary stands and template strands. The heteroduplexes containing a mismatch can be recognized and cleaved by a mismatch endonuclease. The mismatch-containing cleaved heteroduplexes can be removed from the microarray chip using a global buffer exchange. 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.

Claims

1. A method for producing high fidelity oligonucleotides on a solid support, the method comprising:

(a) exposing a plurality of support-bound single-stranded oligonucleotides comprising a predefined sequence to a polymerase enzyme under conditions suitable for a template-dependant synthesis reaction, thereby to produce a plurality of double-stranded oligonucleotides, each of which comprises a support-bound oligonucleotide and a synthesized complementary oligonucleotide;

(b) denaturing the plurality of double-stranded oligonucleotides such that the synthesized oligonucleotides are released from the support-bound oligonucleotides into a solution;

(c) reannealing the synthesized oligonucleotides to the support-bound oligonucleotides, thereby to produce reannealed double-stranded oligonucleotides; and

(d) exposing the reannealed double-stranded oligonucleotides to a mismatch recognizing and cleaving component under conditions suitable for cleavage of reannealed double-stranded oligonucleotides containing a mismatch.

4. A method of assembling nucleic acid polymers comprising the steps of:

a) producing two or more pools of double-stranded high fidelity oligonucleotides according to the method of claim 1;

b) denaturing desirable pools of high fidelity oligonucleotides thereby releasing single-stranded high fidelity oligonucleotides in a solution;

c) combining the desirable pools of single-stranded high fidelity oligonucleotides into a reaction volume; and

d) subjecting the reaction volume to conditions suitable for one or more of hybridization, ligation, and chain extension.

8. The method of any one of claims 1 to 2 wherein the mismatch recognizing and cleaving component comprises a mismatch endonuclease.

9. The method of any one of claims 1 to 2 wherein the mismatch recognizing and cleaving component performs chemical cleavage.

11. The method of any one of claims 1 to 2 wherein the solid support is a microarray.

2. A method for producing high fidelity oligonucleotides on a solid support, the method comprising:

a) synthesizing a first plurality of oligonucleotides in a chain extension reaction using a second plurality of oligonucleotides as templates, wherein the second plurality of oligonucleotides are immobilized on a solid support and comprise an error-containing oligonucleotide having a sequence error at an error-containing position, wherein the chain extension reaction produces a first plurality of duplexes, wherein the first plurality of duplexes comprises homoduplexes;

b) denaturing the first plurality of duplexes to release the first plurality of oligonucleotides from the second plurality of oligonucleotides, wherein the first plurality of oligonucleotides comprise error-free oligonucleotides that are free of error at the error-containing position ;

c) contacting the first plurality of oligonucleotides with the second plurality of oligonucleotides under hybridization conditions to form a second plurality of duplexes, wherein the second plurality of duplexes comprise a mismatch-containing heteroduplex formed between the error-containing oligonucleotide and one of the error-free oligonucleotides; and

d) cleaving off at least a portion of the mismatch-containing heteroduplex by a mismatch recognizing and cleaving component, thereby producing high fidelity double-stranded oligonucleotides.

3. The method of claim 2 further comprising selectively denaturing high fidelity double-stranded oligonucleotides.

5. The method of claim 2 wherein the second plurality of oligonucleotides are chemically synthesized on the solid support and immobilized within one or more features on the solid support.

6. The method of claim 2 wherein the first plurality of oligonucleotides are enzymatically synthesized on the solid support.

7. The method of claim 2 wherein the second plurality of oligonucleotides are attached to at two or more features on the solid support and wherein after step b), one or more of the first plurality of oligonucleotides diffuse away from a point of attachment.

10. The method of claim 2 wherein removing further comprises after cleaving, removing the cleaved duplex by buffer exchange.

12. A method for producing at least one oligonucleotide having a predefined sequence on a solid support, the method comprising:

a. synthesizing on a solid support a first plurality of double-stranded oligonucleotides using a second plurality of oligonucleotides as templates;

b. releasing the first plurality of oligonucleotides from the second plurality of oligonucleotides within an isolated microvolume;

c. contacting the second plurality of oligonucleotides with the first plurality of oligonucleotides under hybridization conditions to form a second plurality of double-stranded oligonucleotides within the isolated microvolume;

d. contacting and cleaving the second plurality of double-stranded oligonucleotides with a mismatch binding agent, wherein the mismatch binding agent selectively binds and cleaves the double-stranded oligonucleotides comprising a mismatch; and

e. removing the double-stranded oligonucleotides comprising the mismatch thereby producing error-free oligonucleotides.

13. The method of claim 12 wherein the mismatch binding agent is a mismatch specific endonuclease.

14. The method of claim 13 wherein the mismatch specific endonuclease cleaves at the region of mismatch.

15. The method of claim 13 wherein the mismatch specific endonuclease is a CEL enzyme.

16. The method of claim 12 wherein the step of releasing the first plurality of oligonucleotides is under denaturing conditions.

17. The method of claim 12 wherein the second plurality of oligonucleotides is bound to a discrete feature of the solid support and wherein the feature is selectively hydrated thereby providing the second plurality of oligonucleotide within the isolated volume.

18. The method of claim 17 wherein the feature is selectively hydrated by spotting a solution comprising a polymerase, dNTPs, a solution promoting primer extension, at least one primer wherein the primer is complementary to a primer binding site on the second plurality of oligonucleotides.

19. The method of claim 12 further releasing error-free oligonucleotides in solution.