Labeling of oligonucleotide probes by multiple-way ligation

A method for producing a labeled, or otherwise modified, oligonucleotide probe, the method comprising the steps of:

• (a) providing a probe-sequence-oligonucleotide comprising (i) a probe sequence comprising a nucleotide sequence complementary to a target nucleic acid and (ii) a predetermined tag nucleotide sequence complementary to a first adaptor nucleotide sequence,
• (b) providing a label-carrier-oligonucleotide comprising at least one labeling moiety, or other functional moiety, wherein the label-carrier-oligonucleotide has a predetermined tag nucleotide sequence complementary to a second adaptor nucleotide sequence,
• (c) providing an adaptor-oligonucleotide, comprising in direct sequence and on a single strand, the first and the second adaptor nucleotide sequence;
• (d) bringing into contact under hybridizing conditions the probe-sequence-oligonucleotide, the label-carrier-oligonucleotide and the adaptor-oligonucleotide, to form a complex, wherein, in the complex, a free/unblocked 3′-OH group is in close spatial proximity to a free/unblocked 5′ phosphate group,
• (e) reacting the complex to form a covalent bond between the 3′-OH group and the 5′-phosphate group using a ligase under ligating conditions, and
• (f) removing the adaptor-oligonucleotide to form the labeled, or otherwise modified, oligonucleotide probe.