Keygene N.v.

René Cornelis Josephus Hogers

Michael Josephus Theresia Van Eijk

Paul Bundock

Michiel Theodoor Jan De Both

Leo Heijnen

Anker Preben SØRENSEN

Ludvik Kevin Wachowski

The invention relates to a kit of parts for detecting one or more polymorphisms in a plurality of target nucleotide sequences in a plurality of samples, wherein the kit of parts comprises (i) a first probe and a second probe, wherein the first probe comprises a first target specific section and a first tag section that is non-complementary to the target nucleotide sequence and that comprises a first universal primer binding site, wherein the second probe comprises a second target specific section and a second tag section that is non-complementary to the target nucleotide sequence; and (ii) a first primer and optionally a second primer wherein the first primer comprises a sample-specific identifier sequence and wherein the first primer can hybridize to the first universal primer binding site.

Sequence based genotyping based on oligonucleotide ligation assays

The invention relates to a method for the detection of a target nucleotide sequence in a sample based on an oligonucleotide ligation assay wherein probes are used that contain (a combination of) sequence-based identifiers that can identify the sample and the target sequence (i.e. locus and/or allele combination) wherein after the ligation step, the ligated probes, or after amplification, the amplified ligated probes, are restricted using restriction enzymes to cut of part of the probes and continue with those parts (identifiers and target sequence) that contain the relevant information in the sequencing step.

Method for de novo whole genome sequencing based on a (sequence-based) physical map of a DNA sample clone bank based on end-sequencing tagged adapter-ligated restriction fragments, in combination with sequencing adapter-ligated restriction fragments of the DNA sample wherein the recognition sequence of the restriction enzyme used in the generation of the physical map is identical to at least part of the recognition sequence of the restriction enzyme used in the generation of the DNA sample.

Restriction enzyme based whole genome sequencing

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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Wageningen, Netherlands

René Cornelis Josephus Hogers