Keygene N.v.

Leo Heijnen

Michael Josephus Theresia Van Eijk

René Cornelis Josephus Hogers

The invention relates to a method for generating, and optionally detecting, an oligonucleotide, comprising at least the steps of: a) providing a first dsDNA; b) ligating the first dsDNA to a second dsDNA, in which said second dsDNA sequence comprises within its sequence at least one recognition site for an IIS restriction endonuclease; so as to provide a ligated dsDNA; d) restricting the ligated dsDNA with the at least one restriction endonuclease of the IIS type so as to obtain at least a first and a second IIS-restricted dsDNA; and optionally comprising the further step of: e) detecting the first and/or second IIS-restricted dsDNA obtained in the restriction step d). Preferably, said method comprises the further step of: c) amplifying the ligated dsDNA obtained in the ligating step b) prior to the restriction step d). The detection step e) preferably comprises the steps of: 1) generating at least one ssDNA from either the first or the second IIS-restricted dsDNA obtained in the restriction step d); 2) detecting the at least one ssDNA generated from either the first or the second IIS-restricted dsDNA; and is more preferably based upon the molecular mass of, and/or the nucleotide sequence of, the fragment(s) obtained in the restriction step d), such as a mass spectroscopy technique, in particular of MALDI-TOF; and/or a chromatography technique, such as high pressure liquid chromatography (HPLC); or a suitable combination thereof, such as Gas Chromatography-Mass Spectroscopy (GC-MS).

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Vianen, Netherlands

Leo Heijnen