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Methods and apparatuses for chip-based DNA error reduction

  • US 9,422,600 B2
  • Filed: 06/20/2011
  • Issued: 08/23/2016
  • Est. Priority Date: 11/25/2009
  • Status: Active Grant
First Claim
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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:

  • (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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