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Methods for high fidelity production of long nucleic acid molecules

  • US 9,499,848 B2
  • Filed: 08/13/2013
  • Issued: 11/22/2016
  • Est. Priority Date: 12/10/2002
  • Status: Active Grant
First Claim
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1. A method for generating a long nucleic acid molecule, the method comprising the steps of:

  • a) providing a plurality of nucleic acids immobilized on a surface, wherein said plurality of nucleic acids comprises nucleic acids having overlapping complementary sequences, wherein said overlapping complementary sequences are capable of hybridizing to each other;

    b) releasing said plurality of nucleic acids into a solution; and

    c) producing the long nucleic acid molecule by the steps of;

    i) forming a plurality of hybridized nucleic acids by hybridizing said overlapping complementary sequences to each other;

    ii) synthesizing a plurality of intermediate-length nucleic acid duplexes by extending said plurality of hybridized nucleic acids;

    iii) forming intermediate-length nucleic acids having overlapping complementary sequences by disassociating said plurality of intermediate-length nucleic acid duplexes, wherein the overlapping complementary sequences in said intermediate-length nucleic acids are capable of hybridizing to each other;

    iv) forming a plurality of intermediate-length hybridized nucleic acids by hybridizing the intermediate-length nucleic acids having overlapping complementary sequences to each other;

    v) synthesizing a plurality of extended intermediate-length nucleic acid duplexes by extending said plurality of intermediate-length hybridized nucleic acids;

    vi) generating a plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule by repeating steps iii) to v) for multiple cycles until said plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule are produced, wherein, in each of the cycles, the plurality of intermediate-length nucleic acid duplexes in step iii) is replaced with the plurality of extended intermediate-length nucleic acid duplexes produced in step v); and

    vii) forming the long nucleic acid molecule by ligating the plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule.

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