5C Technology. Schematic description of Chromosome Conformation Capture Carbon Copy (5C) technology. Illustrated are two strands of DNA in vivo (blue and red double helix), which are bound together by a protein complex (trio of colored spheres). Cells are first crosslinked, which covalently links the protein complex and DNA together. Next, a restriction enzyme is used to cut the DNA at very specific locations throughout the genome. DNA ends are then ligated under dilute conditions in order to promote the formation of DNA junctions between the different strands of DNA linked through a protein complex. The crosslinks are then removed, and the DNA purified, before the annealing of custom 5C primers to individual junctions. A pool of 5C primers is used, represented by the bent lines. Forward primers possess a T7 adaptor (dark green segment), while reverse primers possess a T3c adaptor (purple segment) and a 5' phosphate. All primers have another segment that will bind complementary DNA immediately next to junction sites. Pictured annealing to the single stranded DNA are the red (forward) and blue (reverse) 5C primers. Only primers that are annealed to DNA, are immediately adjacent to one another, and possess a 5' phosphate on the reverse primer will then be ligated by Taq ligase. PCR amplification and labeling is done using the T7 and T3c adaptor sequences, and the resulting library of amplified 5C contacts is hybridized to a custom microarray for detection.