Detection of methylation status

A method of determining methylation status, of at least one nucleotide of interest (NOI) in a non-modified target nucleic acid sequence of interest, wherein said target nucleic acid sequence comprises a target anchor sequence comprising said NOI, said method comprising the steps of

• a) providing an oligonucleotide comprising an anchor sequence (An), wherein the anchor sequence is a sequence at least 50% complementary to said target anchor sequence, wherein the anchor sequence comprises at least one hydrophobic nucleotide (H) positioned between the nucleotide complementary to said NOI and the nucleotide immediately 5′ thereof;
• b) incubating said oligonucleotide with said target nucleic acid sequence of interest at a temperature which is higher than the melting temperature between said oligonucleotide and the target nucleic acid sequence of interest when said NOI is unmethylated,
• c) detecting whether said oligonucleotide anneals to said target nucleic acid sequence of interest,
• thereby determining the methylation status, wherein
• i) said hydrophobic nucleotide (H) has the structure
• 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 an intercalator which is not taking part in Watson-Crick hydrogen bonding;
• and
• Y is a linker moiety linking said nucleotide or nucleotide analogue or backbone monomer unit and said intercalator;
• and
• ii) said oligonucleotide has the structure 5′-An-Lp-St-3′, wherein
• An is the anchor sequence;
• Lp is a loop sequence, which is not complementary to the target nucleic acid sequence of interest, wherein the loop sequence consists of a single nucleic acid sequence capable of forming a protruding structure, or consists of two or more nucleic acid sequences capable of hybridising at least partly to one another to form a complex which is capable of forming a protruding structure;
• St is a starter sequence, capable of hybridizing to a target starter sequence, wherein the target starter sequence is a sequence of the target nucleic acid sequence positioned 5′ to the target anchor sequence.