Chromosome Conformation Analysis

Abstract

Disclosed herein are compositions, methods and kits for analyzing three-dimensional chromatin and/or chromosome conformation. Method are also disclosed for using the methods disclosed herein for diagnosing diseases such as cancer.

1 - 47 . (canceled) 
     
     
         48 . A method of creating a library for chromosomal conformation analysis, comprising:
 a) isolating cells from a biological sample;   b) incubating the cells with a cross-linking agent, thereby cross-linking proteins with DNA and forming a cross-linked product;   c) lysing the cells;   d) digesting the DNA with a restriction endonuclease;   e) filling-in and A-tailing the free ends of the digested DNA;   f) optionally methylating the digested DNA   g) ligating a first half-adaptor to the digested DNA thereby forming a first ligation product and a second half-adaptor to the digested DNA thereby forming a second ligation product, wherein the first and second half-adaptors are different from each other and each half-adaptor comprises a non-palindromic overhang on one end and a T overhang on the other end, wherein the non-palindromic overhang of the first adaptor is complementary to the non-palindromic overhang of the second adaptor, further wherein the first or the second half-adaptor is biotinylated;   h) phosphorylating the ligated half-adaptors;   i) nick ligating the first and second ligation products, thereby forming a third ligation product;   j) reversing the cross-linking;   k) purifying the third ligation product;   l) digesting the third ligation product with a restriction endonuclease;   m) filling-in and/or dA-tailing the free ends of the digested DNA;   n) isolating the digested DNA using streptavidin beads;   o) ligating the digested DNA with sequencing primers;   p) analyzing the DNA.   
     
     
         49 . The method according to  claim 48 , wherein the analyzing step is performed using DNA sequence analysis. 
     
     
         50 . The method according to  claim 49 , wherein the DNA sequencing analysis is next generation sequencing. 
     
     
         51 . The method according to  claim 48 , further comprising the step of incubating the cross-linked product with a cross-linking quencher. 
     
     
         52 . The method according to  claim 48 , wherein the lysing step comprises incubating the cells with one or more detergents. 
     
     
         53 . The method according to  claim 52 , wherein the one or more detergents are selected from the group consisting of anionic detergents and non-ionic detergents. 
     
     
         54 . The method according to  claim 52 , wherein the one or more detergents comprises a combination of one or more anionic detergents and one or more non-ionic detergents. 
     
     
         55 . The method according to  claim 48 , wherein the cross-linking agent comprises formaldehyde. 
     
     
         56 . The method according to  claim 48 , further comprising a control assay to monitor undigested template DNA, wherein the control assay comprises:
 a) a forward primer that hybridizes to a region in the template DNA upstream of the restriction endonuclease cutting site; and   b) a reverse primer that hybridizes to a region in the template DNA downstream of the restriction endonuclease cutting site.   
     
     
         57 . The method according to  claim 48 , further comprising a control assay to monitor the digested template DNA, wherein the control assay comprises:
 a) a bridge oligo comprising a blocked 3′ end, a template DNA binding region and a primer binding region;   b) a forward primer that hybridizes to a region in the 5′ end of the bridge oligo that is upstream of the restriction endonuclease cutting site;   c) a reverse primer that hybridizes to the template DNA that corresponds to a region downstream of the restriction endonuclease cutting site; and   d) optionally an oligonucleotide probe that anneals to a region containing the restriction endonuclease cutting site.   
     
     
         58 . A kit comprising:
 a) a cross-linking agent;   b) a ligation solution;   c) a DNA ligase;   d) a lysis solution;   e) a cross-linking reversing agent.   
     
     
         59 . The kit according to  claim 58 , wherein the cross-linking agent comprises formaldehyde. 
     
     
         60 . The kit according to  claim 58 , wherein the lysis solution comprises one or more detergents. 
     
     
         61 . The kit according to  claim 60 , wherein the one or more detergents are selected from the group consisting of anionic detergents and non-ionic detergents. 
     
     
         62 . The kit according to  claim 60 , wherein the one or more detergents comprises a combination of one or more anionic detergents and one or more non-ionic detergents. 
     
     
         63 . The kit according to  claim 58 , wherein the cross-linking reversing agent comprises a protease. 
     
     
         64 . A composition comprising: a) two or more half-adaptors; b) at least one ligation product; and c) at least one ligase. 
     
     
         65 . The composition according to  claim 64 , further comprising one or more amplification primers. 
     
     
         66 . The composition according to  claim 65 , further comprising a bridge oligo. 
     
     
         67 - 68 . (canceled)