Mapping networks of physical interactions between genomic elements using 5C technology.
Genomic elements separated by large genomic distances can physically interact to mediate long-range gene regulation and other chromosomal processes. Interactions between genomic elements can be detected using the chromosome conformation capture (3C) technology. We recently developed a high-throughput adaptation of 3C, 3C-carbon copy (5C), that is used to measure networks of millions of chromatin interactions in parallel. As in 3C, cells are treated with formaldehyde to cross-link chromatin interactions. The chromatin is solubilized, digested with a restriction enzyme and ligated at low DNA concentration to promote intra-molecular ligation of cross-linked DNA fragments. Ligation products are subsequently purified to generate a 3C library. The 5C technology then employs highly multiplexed ligation-mediated amplification (LMA) to detect and amplify 3C ligation junctions. The resulting 5C library of ligated primers is analyzed using either microarray detection or ultra-high-throughput DNA sequencing. The 5C protocol described here can be completed in 13 d.
Dostie
Josée
J
Program in Gene Function and Expression and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Lazare Research Building, 364 Plantation Street, Room 519, Worcester, Massachusetts 01605-4321, USA.
Dekker
Job
J
D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D023281:Genomics / Q000379:methods*; D008968:Molecular Conformation; D016133:Polymerase Chain Reaction / Q000379:methods
D002843:Chromatin