METHOD FOR THE MANUFACTURE OF NUCLEIC ACID MOLECULES

摘要

The present invention is related to 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 singlestranded overhang, c) ligating the first and the second oligonucleotide via their overhangs generating a first ligation product, d) immobilising the first ligation product 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).