METHOD FOR GENERATING OLIGONUCLEOTIDES, IN PARTICULAR FOR THE DETECTION OF AMPLIFIED RESTRICTION FRAGMENTS OBTAINED USING AFLP3
The invention relates to a method for generating, and optionally detecting, an oligonucleotide, comprising at least the steps of: a) providing a first <i>ds</i>DNA; b) ligating the first <i>ds</i>DNA to a second <i>ds</i>DNA, in which said second <i>ds</i>DNA sequence comprises within its sequence at least one recognition site for an IIS restriction endonuclease; so as to provide a ligated <i>ds</i>DNA; d) restricting the ligated <i>ds</i>DNA with the at least one restriction endonuclease of the IIS type so as to obtain at least a first and a second IIS-restricted <i>ds</i>DNA; and optionally comprising the further step of: e) detecting the first and/or second IIS-restricted <i>ds</i>DNA obtained in the restriction step d). Preferably, said method comprises the further step of: c) amplifying the ligated <i>ds</i>DNA 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 <i>ss</i>DNA from either the first or the second IIS-restricted <i>ds</i>DNA obtained in the restriction step d); 2) detecting the at least one <i>ss</i>DNA generated from either the first or the second IIS-restricted <i>ds</i>DNA; 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).