Chromosome conformation capture

Chromosome conformation capture techniques (often abbreviated to 3C technologies or 3C-based methods) are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome. Such interactions may result from biological functions, such as promoter-enhancer interactions, or from random polymer looping, where undirected physical motion of chromatin causes loci to collide. Interaction frequencies may be analyzed directly, or they may be converted to distances and used to reconstruct 3-D structures.

The chief difference between 3C-based methods is their scope. For example, when using PCR to detect interaction in a 3C experiment, the interactions between two specific fragments are quantified. In contrast, Hi-C quantifies interactions between all possible pairs of fragments simultaneously. Deep sequencing of material produced by 3C also produces genome-wide interactions maps.

Historically, microscopy was the primary method of investigating nuclear organization, which can be dated back to 1590.

All 3C methods start with a similar set of steps, performed on a sample of cells.

First, the cell genomes are cross-linked with formaldehyde, which introduces bonds that "freeze" interactions between genomic loci. Treatment of cells with 1-3% formaldehyde, for 10-30min at room temperature is most common, however, standardization for preventing high protein-DNA cross linking is necessary, as this may negatively affect the efficiency of restriction digestion in the subsequent step. The genome is then cut into fragments with a restriction endonuclease. The size of restriction fragments determines the resolution of interaction mapping. Restriction enzymes (REs) that make cuts on 6bp recognition sequences, such as EcoR1 or HindIII, are used for this purpose, as they cut the genome once every 4000bp, giving ~ 1 million fragments in the human genome. For more precise interaction mapping, a 4bp recognizing RE may also be used. The next step is, proximity based ligation. This takes place at low DNA concentrations or within intact, permeabilized nuclei in the presence of T4 DNA ligase, such that ligation between cross-linked interacting fragments is favored over ligation between fragments that are not cross-linked. Subsequently, interacting loci are quantified by amplifying ligated junctions by PCR methods.

The chromosome conformation capture (3C) experiment quantifies interactions between a single pair of genomic loci. For example, 3C can be used to test a candidate promoter-enhancer interaction. Ligated fragments are detected using PCR with known primers. That is why this technique requires the prior knowledge of the interacting regions.

Chromosome conformation capture-on-chip (4C) (also known as circular chromosome conformation capture) captures interactions between one locus and all other genomic loci. It involves a second ligation step, to create self-circularized DNA fragments, which are used to perform inverse PCR. Inverse PCR allows the known sequence to be used to amplify the unknown sequence ligated to it. In contrast to 3C and 5C, the 4C technique does not require the prior knowledge of both interacting chromosomal regions. Results obtained using 4C are highly reproducible with most of the interactions that are detected between regions proximal to one another. On a single microarray, approximately a million interactions can be analyzed. ^{[citation needed]}

Chromosome conformation capture carbon copy (5C) detects interactions between all restriction fragments within a given region, with this region's size typically no greater than a megabase. This is done by ligating universal primers to all fragments. However, 5C has relatively low coverage. The 5C technique overcomes the junctional problems at the intramolecular ligation step and is useful for constructing complex interactions of specific loci of interest. This approach is unsuitable for conducting genome-wide complex interactions since that will require millions of 5C primers to be used.^{[citation needed]}