Application of epigenetic chromsomal interactions in cancer diagnostics

A method for quantitatively detecting a ligated sequence with high sensitivity, which ligated sequence corresponds to an epigenetic chromosome interaction, wherein the method uses a probe which is detectable upon activation during a PCR reaction,

• wherein the ligated sequence comprises sequences from each of the two chromosome regions that come together in an epigenetic chromosome interaction and:
• (a) the method comprises firstly making the ligated sequence by a process comprising the steps of:
• (i) in vitro cross-linking of the two chromosome regions of the epigenetic chromosome interaction to form cross-linked nucleic acid sequence,
• (ii) subjecting said cross-linked nucleic acid sequence to restriction digestion cleavage with an enzyme,
• (iii) ligating the ends of the cross-linked cleaved nucleic acid to form the ligated sequence;
• and
• (b) wherein the method secondly comprises contacting the ligated sequence with the probe during the PCR reaction, and detecting the extent of activation of the probe, wherein said quantitatively detecting comprises specific detection of the ligated sequence by quantitative PCR (qPCR) which uses primers capable of amplifying the ligated sequence and a said probe which binds specifically to the ligation site during the PCR reaction, and
• wherein the probe comprises:
• a fluorophore covalently attached to the 5′ end of the probe, and
• a quencher covalently attached to the 3′ end of the probe,
• wherein specific binding of the probe to the ligation site means that non-ligated sequences are not detected.