Fig. 3. Two methods used to illustrate looping. (A) 3C. A loop is shown, tethered by transcription units a and c to a protein cluster (small pink circle). The 3C method involves fixation to covalently cross-link DNA sequences lying next to each other (usually through DNA:protein:DNA links shown as the red line), before removing unlinked proteins, cutting DNA, dilution and ligation. Dilution favours intramolecular ligation, so the ends of two different DNA molecules in one DNA:protein:DNA complex are joined together more frequently than those in different molecules/complexes. Then, two DNA sequences initially lying together in 3D space are often ligated together (i.e. a with c, but not b), and (after reversing crosslinks) their juxtaposition is detected using PCR. (B) RNA TRAP. Only the region around transcription units a and c is illustrated. This method also involves fixation to preserve molecular contacts, in situ hybridization of a tagged intron probe to specific primary transcripts at a target transcription site (i.e. a), immunolabelling to bring peroxidase activity to the tagged probe, and reaction with biotintyramide to generate free-radicals that biotinylate proteins in the vicinity. Then DNA sequences initially lying near the target site in 3D space (c, but not b) co-purify with biotinylated chromatin and can be detected by PCR.