Methods and apparatuses for chip-based DNA error reduction
First Claim
1. A method for producing a population of double-stranded oligonucleotides having improved fidelity on a solid support, the method comprising:
- (a) contacting, on a solid support, a plurality of support-bound single-stranded oligonucleotides with a solution comprising a primer and a polymerase enzyme under conditions suitable for a template-dependent synthesis reaction, thereby producing a plurality of double-stranded oligonucleotides comprising synthesized complementary oligonucleotides base paired with the support-bound single-stranded oligonucleotides, wherein the support-bound single-stranded oligonucleotides are bound on the solid support at their 3′
ends and comprise error-free oligonucleotides and error-containing oligonucleotides;
(b) denaturing the plurality of double-stranded oligonucleotides such that the synthesized complementary oligonucleotides are released in one or more droplets;
(c) reannealing the synthesized complementary oligonucleotides in the one or more droplets to the support-bound single-stranded oligonucleotides, thereby producing reannealed double-stranded oligonucleotides comprising homoduplexes and heteroduplexes, wherein each of the heteroduplexes comprises a mismatch;
(d) exposing the reannealed double-stranded oligonucleotides to a mismatch recognizing and cleaving component under conditions suitable for cleavage of the heteroduplexes, thereby cleaving at least a portion of the heteroduplexes; and
(e) removing at least a portion of the cleaved heteroduplexes, thereby producing the population of double-stranded oligonucleotides having improved fidelity on the solid support.
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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.
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Citations
21 Claims
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1. A method for producing a population of double-stranded oligonucleotides having improved fidelity on a solid support, the method comprising:
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(a) contacting, on a solid support, a plurality of support-bound single-stranded oligonucleotides with a solution comprising a primer and a polymerase enzyme under conditions suitable for a template-dependent synthesis reaction, thereby producing a plurality of double-stranded oligonucleotides comprising synthesized complementary oligonucleotides base paired with the support-bound single-stranded oligonucleotides, wherein the support-bound single-stranded oligonucleotides are bound on the solid support at their 3′
ends and comprise error-free oligonucleotides and error-containing oligonucleotides;(b) denaturing the plurality of double-stranded oligonucleotides such that the synthesized complementary oligonucleotides are released in one or more droplets; (c) reannealing the synthesized complementary oligonucleotides in the one or more droplets to the support-bound single-stranded oligonucleotides, thereby producing reannealed double-stranded oligonucleotides comprising homoduplexes and heteroduplexes, wherein each of the heteroduplexes comprises a mismatch; (d) exposing the reannealed double-stranded oligonucleotides to a mismatch recognizing and cleaving component under conditions suitable for cleavage of the heteroduplexes, thereby cleaving at least a portion of the heteroduplexes; and (e) removing at least a portion of the cleaved heteroduplexes, thereby producing the population of double-stranded oligonucleotides having improved fidelity on the solid support. - View Dependent Claims (3, 4, 8, 9, 10, 11, 12, 13)
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2. A method for producing a population of double-stranded oligonucleotides having improved fidelity on a solid support, the method comprising:
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(a) synthesizing a first plurality of oligonucleotides in a chain extension reaction using a second plurality of support-bound oligonucleotides as templates in the presence of a solution comprising a primer, wherein the second plurality of oligonucleotides is bound on a solid support at their 3′
ends and comprise an error-containing oligonucleotide having a sequence error at an error-containing position, thereby producing a first plurality of duplexes, wherein the first plurality of duplexes comprises homoduplexes;(b) denaturing the first plurality of duplexes, thereby releasing the first plurality of oligonucleotides in one or more droplets, wherein the first plurality of oligonucleotides comprise error-free oligonucleotides that are free of error at a position corresponding to the error-containing position of the error-containing oligonucleotide in the second plurality of oligonucleotides; (c) contacting the first plurality of oligonucleotides in the one or more droplets with the second plurality of oligonucleotides under hybridization conditions such that a second plurality of duplexes is formed, wherein the second plurality of duplexes comprise a mismatch-containing heteroduplex formed between the error-containing oligonucleotide and one of the error-free oligonucleotides; (d) cleaving the mismatch-containing heteroduplex by a mismatch recognizing and cleaving component; and (e) removing the mismatch-containing heteroduplex, thereby producing the population of double-stranded oligonucleotides having improved fidelity on the solid support. - View Dependent Claims (5, 6, 7)
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14. A method for producing at least one support-bound error-free oligonucleotide having a predefined sequence on a solid support, the method comprising:
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(a) synthesizing a first plurality of oligonucleotides on a solid support using a second plurality of support-bound oligonucleotides as templates in the presence of at least one primer, wherein the second plurality of support-bound oligonucleotides is bound on the solid support at their 3′
ends,wherein the at least one primer is complementary to a primer binding site on the second plurality of oligonucleotides, wherein each of the second plurality of oligonucleotides has a predefined sequence, and wherein at least one of the second plurality of support-bound oligonucleotides comprises a sequence error; (b) releasing the first plurality of oligonucleotides in one or more droplets; (c) contacting the second plurality of support-bound oligonucleotides with the first plurality of oligonucleotides in the one or more droplets under hybridization conditions such that a plurality of double-stranded oligonucleotides is formed, wherein the plurality of double-stranded oligonucleotides comprises a double-stranded oligonucleotide having a mismatch with the sequence error; (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 oligonucleotide having the mismatch; and (e) removing the double-stranded oligonucleotide having the mismatch, thereby producing the at least one support-bound error-free oligonucleotide having the predefined sequence on the solid support. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21)
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Specification