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Solid phase enzymatic assembly of polynucleotides

  • US 6,479,262 B1
  • Filed: 05/16/2000
  • Issued: 11/12/2002
  • Est. Priority Date: 05/16/2000
  • Status: Expired due to Term
First Claim
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1. A method of preparing a polynucleotide having at least 200 nucleotides and a predetermined nucleotide sequence comprising the steps:

  • a) providing a solid support;

    b) providing a plurality of oligonucleotides, wherein the combination of the nucleotide sequences of said oligonucleotides comprises the nucleotide sequence of said polynucleotide;

    c) contacting said solid support with the 3′

    terminus of a first oligonucleotide from said plurality of oligonucleotides to form a tethered oligonucleotide;

    d) ligating the 3′

    terminus of another oligonucleotide from said plurality of oligonucleotides to the 5′

    terminus of said tethered oligonucleotide, wherein said ligating is carried out in the presence of RNA ligase or ribozyme;

    e) phosphorylating the 5′

    terminus of said another oligonucleotide; and

    f) repeating steps d) and e) until said polynucleotide is prepared.

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