This is an alternative protocol for 3C that has been adopted by the author's lab. Much of it is identical to the previous version (PROT5). The major differences are in the amounts of DNA used at different steps. We reliably get the same results as previously, but also a greater yield of 3C material. The original yields are ample for real-time PCR analysis, but greater yields are required if the 3C material is going to be processed further.
The 3C (Chromosome Conformation Capture) technique generates a population average measurement of juxtaposition frequency between any two genomic loci, thus providing information on their relative proximity in the nucleus (Dekker et al., 2002). Cells are fixed with formaldehyde which forms DNA-protein and protein-protein cross-links between regions of the genome in proximity. Subsequent restriction enzyme digestion and intra-molecular ligation produces novel junctions between restriction fragments in proximity in the nucleus. Novel ligation products can be detected by PCR. We adapted the 3C assay (Dekker et al., 2002) to determine the conformation of mouse chromosome 7 and in particular the co-localization of actively transcribed genes in transcription factories (Osborne et al., 2004). The 3C assay can also be used to reveal proximity between active genes and distal genomic elements (Tolhuis et al., 2002).
An important consideration in the interpretation of 3C data is the understanding that not all pairs of restriction fragments that provide a positive result (i.e. generate a novel PCR product) are necessarily engaged in a functional interaction in the nucleus. For example, compare results of Tolhuis et al., 2002, with Carter et al., 2002 in which the higher order structure of the mouse hbb locus was assayed by two different methods, 3C and RNA TRAP. Clearly, distal fragments can be cross-linked by formaldehyde simply because they are near each other in the nucleus, and presumably can "bump into" each other during the fixation process (Osborne et al., 2004). Therefore fixation conditions are critical in the 3C assay since increased fixation leads to greater cross-linking resulting in the detection of chromatin fragments that may be in proximity in the nucleus but not necessarily engaged in a specific intermolecular interaction with implied function.
Laboratory of Chromatin and Gene Expression - The Babraham Institute - Cambridge, UK