Oligonucleotides comprising signalling pairs and hydrophobic nucleotides, stemless beacons, for detection of nucleic acids, methylation status and mutants of nucleic acids

A method for determining hybridisation comprising the steps of

• a) providing an oligonucleotide analogue comprising a consecutive sequence of n nucleotides and/or nucleotide analogues and at least two hydrophobic nucleotides, wherein said oligonucleotide analogue is covalently linked to a signalling pair consisting of a two part system,
• wherein the hydrophobic nucleotide has the general structure
• X-Y-Q
• wherein
• X is a nucleotide or nucleotide analogue or a backbone monomer unit capable of being incorporated into the backbone of a nucleic acid or nucleic acid analogue,
• Q is a hydrophobic molecule which is not taking part in specific Watson-Crick hydrogen bonding;
• and
• Y is a linker moiety linking said nucleotide or nucleotide analogue or backbone monomer unit and said hydrophobic molecule;
• and
• wherein n is an integer of at least 6;
• and
• wherein the two parts of the signalling pair are in closer proximity, when said oligonucleotide analogue is not hybridised to a target sequence than when said oligonucleotide analogue is hybridised to a target sequence;
• and
• b) providing a mixture of nucleic acids potentially comprising a target sequence for said oligonucleotide analogue;
• and
• c) incubating said nucleic acids and said oligonucleotide analogue under conditions allowing for hybridisation;
• and
• d) detecting hybridisation by determining whether the parts of the signalling pair are in close proximity;
• e) thereby determining hybridisation.

A method for amplification of a target sequence, said method comprising the steps of:

• a) providing a mixture of nucleic acids optionally comprising said target sequence;
• and
• b) providing a set of primers, an amplification buffer comprising an amplification enzyme, and an oligonucleotide or oligonucleotide analogue, wherein said primers and said oligonucleotide or oligonucleotide analogue is capable of hybridizing to said target sequence or a sequence complementary thereto, with the proviso that when the amplification enzyme comprises nuclease activity then the oligonucleotide or oligonucleotide analogue is nuclease resistant;
• and
• c) incubating the primers and oligonucleotide or oligonucleotide analogue with the mixture of nucleic acids under conditions allowing for hybridization;
• and
• d) detecting hybridisation;
• and
• e) gradually increase the temperature while detecting hybridisation at certain intervals until the desired extension temperature is reached, and
• f) finish the elongation reaction and denature the formed amplicon, and repeat step c) to f) until a desired amplification has taken place.