Methods and kits for methylation detection

A method for determining the degree of methylation of a target nucleotide comprising,

• (a) reacting (1) a target sequence with (2) a first cleavage probe set comprising (i) a first cleavage probe that can hybridize with the target sequence and (ii) a second cleavage probe that (a) can hybridize with the target sequence downstream of the first cleavage probe and (b) contains a flap portion, under effective conditions for the first and second cleavage probes of the first cleavage probe set to hybridize with the target sequence, forming a first hybridization complex;
• (b) cleaving the flap portion of the second cleavage probe in the first hybridization complex to generate a cleaved flap and form a second hybridization complex comprising (1) the target sequence, (2) the first cleavage probe, and (3) an annealed fragment of the second cleavage probe having a 5′-terminal nucleotide located adjacent to the 3′-end of the annealed first cleavage probe;
• (c) ligating the first cleavage probe to the annealed fragment of the second cleavage probe to generate a first ligation product and form a third hybridization complex comprising the target sequence and the first ligation product;
• (d) denaturing the third hybridization complex;
• (e) performing one or more additional cycles of steps (a) through (c) and optionally, step (d);
• and
• (f) determining the degree of methylation of the target nucleotide.

A method for determining the degree of methylation of a target nucleotide, comprising:

• a step for interrogating the target nucleotide;
• a step for generating a cleaved flap;
• a step for generating a ligation product;
• and
• a step for determining the degree of methylation of the target nucleotide.