Patent AT-E400650-T1: [Translated] METHOD FOR PRODUCING NUCLEIC ACID MOLECULES

Manufacturing a nucleic acid molecule comprising employing at least partially double-stranded oligonucleotide comprising a modification allowing the oligonucleotide to be coupled to a surface, a recognition site for a first type IIS restriction enzyme that cuts outside its recognition site, and a single-stranded overhang, is new. Manufacturing a nucleic acid molecule comprises: (a) providing a first at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled to a surface, comprises a recognition site for a first type IIS restriction enzyme that cuts outside its recognition site, and comprises a single-stranded overhang; (b) providing a second at least partially double-stranded oligonucleotide, which comprises a recognition site, its part or its complementary sequence, for a second type IIS restriction enzyme which cuts outside its recognition site, and comprises a single-stranded overhang; (c) ligating the first and the second oligonucleotide via their overhangs to generate a first ligation product; (d) immobilizing the first ligation product to the surface via the modification; (e) cutting the immobilized ligation product with the first type IIS restriction enzyme to release 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, a recognition site for a further type IIS restriction enzyme that cuts outside its recognition site, and a single-stranded overhang, and ligating the elongated second oligonucleotide and the further at least partially double-stranded oligonucleotide via their overhangs to form a further ligation product; (g) immobilizing the further ligation product to a surface via the modification; (h) cutting the further ligation product with the further type IIS restriction enzyme to release an elongated oligonucleotide having an overhang; and (i) optionally, repeating steps (f)-(h).