Application of epigenetic chromosomal interactions in cancer diagnostics

Abstract

The invention provides a method of determining the epigenetic chromosome interactions which are relevant to a prognostic companion epigenetic test for cancer.

1 .- 15 . (canceled) 
     
     
         16 . A method for quantitatively detecting a ligated sequence which is relevant to a chromosome interaction using a probe which is detectable upon activation during a PCR reaction, 
       wherein said ligated sequence comprises sequences from two chromosome regions that come together in an epigenetic chromosome interaction, 
       wherein said method comprises contacting the ligated sequence with the probe during a PCR reaction, and detecting the extent of activation of the probe, and wherein said probe binds the ligation site. 
     
     
         17 . A method according to  claim 16  wherein said ligated product or sequence has a curvature propensity peak score of at least 5° per helical turn. 
     
     
         18 . A method according to  claim 17  wherein said curvature propensity score per helical turn is calculated for up to 20 to 400 bases upstream and/or downstream of the ligation site. 
     
     
         19 - 21 . (canceled) 
     
     
         22 . The method of  claim 16  wherein the ligated sequence is formed by a method comprising the steps of:
 (i) in vitro cross-linking of the two chromosome regions to form cross-linked nucleic acid; 
 (ii) subjecting said cross-linked acid to restriction digestion cleavage with an enzyme; and 
 (iii) ligating said cross-linked cleaved nucleic acid ends to form the ligated sequence. 
 
     
     
         23 . The method of  claim 16  wherein the ligation site of said ligated sequence comprises the restriction enzyme recognition sequence of the restriction enzyme used to cut the crosslinked nucleic acid, and preferably said restriction enzyme is Taq1. 
     
     
         24 . The method of  claim 16  wherein detection of the ligated sequence allows a determination of an epigenetic chromosome interactions which is specific to a subgroup in a population. 
     
     
         25 . The method of  claim 16  wherein the subgroup is a subgroup of the human population. 
     
     
         26 . The method of  claim 16  wherein said ligated sequence comprises a nucleic acid sequence of length:
 10 to 1000 nucleotide bases, 
 10 to 800 nucleotide bases, 
 10 to 500 nucleotide bases, 
 10 to 100 nucleotide bases, 
 10 to 400 nucleotide bases, 
 10 to 500 nucleotide bases, 
 200 to 600 nucleotide bases, 
 200 to 800 nucleotide bases, or 
 200 to 1000 nucleotide bases. 
 
     
     
         27 . The method of  claim 16  wherein said detecting comprises specific detection of the ligated sequence by quantitative PCR (qPCR) which uses primers capable of amplifying the ligated sequence and a probe which binds the ligation site during the PCR reaction, wherein said probe comprises sequence which is complementary to sequence from each of the chromosome regions that have come together in the chromosome interaction. 
     
     
         28 . The method of  claim 27  wherein said probe comprises:
 an oligonucleotide which specifically binds to said ligated product, and/or 
 a fluorophore covalently attached to the 5′ end of the oligonucleotide, and/or 
 a quencher covalently attached to the 3′ end of the oligonucleotide, and 
 optionally said fluorophore is selected from HEX, Texas Red and FAM. 
 
     
     
         29 . The method of  claim 27  wherein said probe comprises a nucleic acid sequence of length 10 to 40 nucleotide bases, preferably a length of 20 to 30 nucleotide bases. 
     
     
         30 . The method of  claim 16  wherein said sequences from two chromosome regions that come together in an epigenetic chromosome interaction lie at least 1000 base pairs apart or at least 10,000 base pairs apart or at least 100,000 base pairs apart in the chromosome. 
     
     
         31 . The method of  claim 16  which is carried out on sample from an individual and which further comprises administering a therapeutic agent to the individual, wherein said individual has been identified as being in need of said therapeutic agent by the method of  claim 16 .