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De novo enzymatic production of nucleic acid molecules

  • US 8,092,991 B2
  • Filed: 01/22/2005
  • Issued: 01/10/2012
  • Est. Priority Date: 01/23/2004
  • Status: Active Grant
First Claim
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1. A method for the manufacture of a nucleic acid molecule comprising the following steps:

  • (a) providing a first at least partially double-stranded oligonucleotide, whereby the oligonucleotide comprises a first single-stranded overhang, and a first modification allowing the oligonucleotide to be immobilised to a surface, wherein the first modification comprises a second single-stranded nucleotide overhang, wherein the first oligonucleotide is a first part of a nucleic acid to be manufactured,(b) providing a second at least partially double-stranded oligonucleotide, which comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, a second modification allowing the oligonucleotide to be coupled to a surface and a single-stranded overhang, wherein the second oligonucleotide is a second part of a nucleic acid to be manufactured,(c) ligating the first oligonucleotide and the second oligonucleotide via the first single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide, generating a first ligation product, whereby the first ligation product comprises the first modification allowing the first ligation product to be immobilised to a surface, wherein the first modification of the first ligation product is the second single-stranded nucleotide overhang of the first oligonucleotide,(d) cutting the first ligation product with the first type IIS restriction enzyme thus releasingan elongated first at least partially double-stranded oligonucleotide having a first and a second single-stranded overhang, whereby the first single-stranded overhang is generated through the cutting of the restriction enzyme and whereby the second single-stranded overhang is the second single-stranded nucleotide overhang of the first at least partially double-stranded oligonucleotide of step (a), anda truncated second at least partially double-stranded oligonucleotide;

    (e) immobilising the truncated second at least partially double stranded oligonucleotide of step d), the unreacted second at least partially double-stranded oligonucleotide and/or the uncut first ligation product via the second modification to a surface;

    (f) repeating steps (a) to (e) at least once, whereby the elongated first at least partially double-stranded oligonucleotide of step (d) serves as the first at least partially double-stranded oligonucleotide in step (a), and is further elongated.

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