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Methods and Devices for High Fidelity Polynucleotide Synthesis

  • US 20140309119A1
  • Filed: 06/27/2014
  • Published: 10/16/2014
  • Est. Priority Date: 08/27/2008
  • Status: Active Grant
First Claim
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1. A method for assembling a polynucleotide having a predefined sequence from a plurality of different oligonucleotides, the method comprising:

  • a) providing a plurality of single-stranded template oligonucleotides on a support, wherein each of the plurality of template oligonucleotides comprises a predefined sequence and includes a primer binding site;

    b) generating a complementary oligonucleotide for each of the plurality of template oligonucleotides by enzyme-catalyzed synthesis within a primary droplet, thereby producing a plurality of double-stranded oligonucleotides;

    c) releasing the complementary oligonucleotide from the double-stranded oligonucleotides into the primary droplet;

    d) combining at least a first and second primary droplets, thereby forming a secondary droplet, wherein the first primary droplet includes a released oligonucleotide that comprises a portion that is complementary to a portion of a released or template oligonucleotide from the second primary droplet; and

    e) exposing the secondary droplet to conditions suitable for hybridization and ligation, polymerase extension, or polymerase extension and ligation to assemble a double-stranded polynucleotide having a predefined sequence.

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