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Assembly of High Fidelity Polynucleotides

  • US 20130059761A1
  • Filed: 01/06/2011
  • Published: 03/07/2013
  • Est. Priority Date: 01/07/2010
  • Status: Active Grant
First Claim
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1. A method for producing at least one polynucleotide having a predefined sequence, the method comprising:

  • a) providing at least a first and a second plurality of support-bound single-stranded oligonucleotides, wherein each first and second plurality of support-bound oligonucleotides has a predefined sequence and is bound to a discrete feature of the support, each first plurality of support-bound oligonucleotides comprising a sequence region at its 5′

    end that is the same as a sequence region of a 3′

    end of the second plurality of support-bound oligonucleotides;

    b) providing a plurality of first input single-stranded oligonucleotides, wherein the 3′

    end of the plurality of the first input oligonucleotide is complementary to the 3′

    end of the first plurality of support-bound oligonucleotides;

    c) hybridizing the plurality of first input oligonucleotides to the first plurality of support-bound oligonucleotides at a first feature;

    d) generating a first plurality of complementary oligonucleotides in a chain extension reaction, thereby forming an extension product duplex;

    e) dissociating the extension product duplex, thereby producing a first plurality of complementary oligonucleotides;

    f) transferring the first plurality of complementary oligonucleotides from the first feature to a second feature, thereby bringing into contact the first plurality of complementary oligonucleotides to the second plurality of support-bound oligonucleotides;

    g) annealing the first plurality of complementary oligonucleotides to the second plurality of support-bound single-stranded oligonucleotides at the second feature, wherein the annealing of the first plurality of complementary oligonucleotides to the second plurality of support-bound oligonucleotides serves as a primer for extension of the first plurality of complementary oligonucleotides, thereby producing the polynucleotide; and

    h) optionally amplifying the polynucleotide.

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