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Method for the manufacture of nucleic acid molecules

  • US 7,695,906 B2
  • Filed: 10/17/2003
  • Issued: 04/13/2010
  • Est. Priority Date: 10/18/2002
  • Status: Active Grant
First Claim
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1. A method for the manufacture of a nucleic acid molecule comprising the steps of:

  • (a) providing a first at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide comprises a single-stranded overhang;

    (b) providing a second at least partially double-stranded oligonucleotide whereby the oligonucleotide comprises a recognition site or a part thereof or a sequence which is complementary thereto, for a second type IIS restriction enzyme which cuts outside its recognition site, and which second oligonucleotide comprises a single-stranded overhang;

    (c) ligating the first and the second oligonucleotide via their overhangs generating a first ligation product, wherein the first and second oligonucleotides are not attached to a surface during the ligation;

    (d) immobilising the first ligation product of step (c) to the surface via the modification;

    (e) cutting the immobilised ligation product with the first type IIS restriction enzyme thus releasing an elongated oligonucleotide having an overhang;

    (f) combining the elongated oligonucleotide with a further at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled, to a surface, whereby the further oligonucleotide comprises a recognition site for a further type IIS restriction enzyme which cuts outside its recognition site and which oligonucleotide comprises a single-stranded overhang, and ligating the elongated second oligonucleotide and the further at least partially double-stranded oligonucleotide via their overhangs forming a further ligation product;

    (g) immobilising the further ligation product to a surface via the modification;

    (h) cutting the further ligation product with the further type IIS restriction enzyme releasing an elongated oligonucleotide having an overhang; and

    (i) optionally, repeating steps f) to h).

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