Chromosome conformation analysis
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. A method of determining chromosome conformation 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 in a lysing buffer comprising a combination of one or more anionic detergents and one or more non-ionic detergents;
d) digesting the DNA with a restriction endonuclease;
e) ligating the digested DNA thereby creating a ligation product;
f) reversing the cross-linking;
g) purifying the ligation product;
h) analyzing the ligation product; and
i) determining the chromatin conformation.
2. The method according to claim 1 , wherein the cross-linking reagent comprises formaldehyde.
3. The method according to claim 1 , further comprising the step of incubating the cross-linked product with a cross-linking quencher.
4. The method of claim 1 , wherein the purifying step comprises
a) incubating the ligation product with magnetic beads;
b) collecting the ligation product bound to the magnetic beads; and
c) eluting the bound ligation product from the magnetic beads.
5. The method according to claim 1 , wherein the analyzing step is performed using the polymerase chain reaction.
6. The method according to claim 1 , 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.
7. The method according to claim 1 , 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.