Abstract
INTRODUCTIONChromosome conformation capture (3C) is a technique used to detect the spatial organization of chromosomal DNA in fixed cells. DNA sequences in spatial proximity in the nucleus or engaged in physical interactions (such as those between genes and regulatory elements) can be assessed quantitatively to provide a measure that potentially reflects their frequency of association and/or their proximity. 3C can be used to study long-range interactions between DNA sequences on the same chromosome (intrachromosomal) or between different chromosomes (interchromosomal). Briefly, chromatin fragments in proximity are fixed with formaldehyde, followed by digestion with restriction enzymes, nuclear lysis, dilution of the cross-linked complexes, and intramolecular ligation. The cross-links are subsequently removed and the DNA is purified. Polymerase chain reaction (PCR) is then used to amplify DNA fragments containing novel ligation junctions using primers specific for a pair of DNA sequences under investigation. Here we describe a detailed 3C protocol and experimental controls that can be applied to investigate the nuclear juxtaposition of any two genomic regions, in cis or trans.
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