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Method of generating long nucleic acid molecules of defined sequence

  • US 20040241732A1
  • Filed: 04/07/2004
  • Published: 12/02/2004
  • Est. Priority Date: 04/11/2003
  • Status: Abandoned Application
First Claim
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1. A polymerase extension method for generating a single-stranded nucleic acid molecule, the method comprising:

  • a) combining in a mixture under conditions suitable for polymerase extension reaction, (i) a polymerase, (ii) an initial polynucleotide comprising a 5′

    portion and a 3′

    portion, wherein the polynucleotide forms the nucleic acid molecule 5′

    end; and

    (iii) one or more overlapping template oligonucleotides each having a 5′

    template portion, and a 3′

    portion wherein the 3′

    portion of each template oligonucleotide is sufficiently complementary to the 3′

    portion of the initial polynucleotide or to a 3′

    portion of an extension polynucleotide formed by a polymerase extension reaction, to hybridize to the initial polynucleotide or to an extension polynucleotide wherein the initial polynucleotide and each of the one or more overlapping template oligonucleotides consist of not more than 100 contiguous nucleotides;

    b) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized;

    c) subjecting the mixture to denaturing conditions to separate extension polynucleotide and template oligonucleotide;

    d) repeating b) and c).

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