Enzymatic production de novo nucleic acid molecules.

摘要

Process for manufacturing a nucleic acid molecule, comprising the steps of a) providing an at least partially double-stranded first oligonucleotide comprises a recognition site for a first restriction enzyme type IIS which cuts outside its recognition site and said oligonucleotide comprising a single-stranded overhang; b) providing a second oligonucleotide at least partially double-stranded comprising a modification allowing the oligonucleotide coupled to a surface, whereby the oligonucleotide further comprises a recognition site for a second restriction enzyme type IIS that cuts outside its site recognition and said second oligonucleotide has a single-stranded overhang; c) ligating the first and the second oligonucleotide via their outgoing generating a first ligation product; d) immobilising the first ligation product on a surface via the modification provided by the second oligonucleotide; e) cutting the immobilized first ligation product with the second restriction enzyme type IIS thereby releasing a first elongated oligonucleotide having an overhang and a second oligonucleotide shortened, which remains attached to the surface; f) providing a further at least partially double-stranded oligonucleotide having a modification allowing the further oligonucleotide coupled specifically to a surface, whereby the oligonucleotide contains a recognition site for a second or further restriction enzyme type IIS and a single-stranded overhang that is complementary to the overhang of the elongated first oligonucleotide; g) ligating the further at least partially double-stranded oligonucleotide with the elongated first oligonucleotide via their outgoing I generating a ligation product of second level; h) cutting the second ligation product with the second level or additional restriction enzyme type IIS thereby generating an oligonucleotide elongated second level having a projection and an additional oligonucleotide shortened; i) immobilising the shortened further oligonucleotide; j) repeating steps f) to i) at least once, generating in step g) a ligation product of higher level, whereby in the last repetition the further oligonucleotide incoming comprises a recognition site for a restriction enzyme IIS type that produces upon cleavage identical in length to the single-stranded overhang overhang generated by the first restriction enzyme specific for the type IIS first oligonucleotide and steps h) and i) are replaced with k) and l) stages; k) immobilising the ligation product level by modification provided by the further oligonucleotide; l) cutting the ligation product level with the restriction enzyme additional IIS type, leaving the part of the nucleic acid to be manufactured attached to the first oligonucleotide, which is preferably released into the supernatant and more preferably allowing its transfer to a new vessel reaction.