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Chromosome conformation capture (3C) is used to quantify physical DNA contacts in vivo at high resolution. 3C was first used in yeast to map the spatial chromatin organization of chromosome III, and in higher eukaryotes to demonstrate that genomic DNA elements regulate target genes by physically interacting with them. 3C has been widely adopted for small-scale analysis of functional chromatin interactions along (cis) or between (trans) chromosomes. For larger-scale applications, chromosome conformation capture carbon copy (5C) combines 3C with ligation-mediated amplification (LMA) to simultaneously quantify hundreds of thousands of physical DNA contacts by microarray or ultra-high-throughput DNA sequencing. 5C allows the mapping of extensive networks of physical interactions among large sets of genomic elements throughout the genome. Such networks can provide important biological insights, e.g., by identifying relationships between regulatory elements and their target genes. This unit describes 5C for large-scale analysis of cis- and trans-chromatin interactions in mammalian cells.

Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross-linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells.