×

Computer-directed assembly of a polynucleotide encoding a target polypeptide

  • US 20030138782A1
  • Filed: 01/18/2002
  • Published: 07/24/2003
  • Est. Priority Date: 01/19/2001
  • Status: Abandoned Application
First Claim
Patent Images

1. A method of synthesizing a target polynucleotide comprising:

  • a) providing a target polynucleotide sequence;

    b) identifying at least one initiating polynucleotide present in the target polynucleotide of a), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′

    overhang and a 3′

    overhang;

    c) identifying a second polynucleotide present in the target polynucleotide of a), wherein the second polynucleotide is contiguous with the initiating polynucleotide and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′

    overhang, a 3′

    overhang, or a 5′

    overhang and a 3′

    overhang, wherein at least one overhang of the second polynucleotide is complementary to at least one overhang of the initiating polynucleotide;

    d) identifying a third polynucleotide present in the target polynucleotide of a), wherein the third polynucleotide is contiguous with the initiating sequence and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′

    overhang, a 3′

    overhang, or a 5′

    overhang and a 3′

    overhang, wherein at least one overhang of the third polynucleotide is complementary to at least one overhang of the initiating polynucleotide which is not complementary to an overhang of the second polynucleotide;

    e) contacting the initiating polynucleotide of b) with the second polynucleotide of c) and the third polynucleotide of d) under conditions and for such time suitable for annealing, the contacting resulting in a contiguous double-stranded polynucleotide, wherein the initiating sequence is extended bi-directionally;

    f) in the absence of primer extension, optionally contacting the mixture of e) with a ligase under conditions suitable for ligation; and

    g) optionally repeating b) through f) to sequentially add double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized.

View all claims
  • 1 Assignment
Timeline View
Assignment View
    ×
    ×