Patent ID: 8092991
Filing Date: 2012-01-10
Classification: C12N,C12P

Abstract:
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 releasing an 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), and a 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.