Chromosome Conformation Capture - Chromosome Conformation Capture (3C)

Chromosome Conformation Capture (3C)

The basic 3C technique has five experimental steps:

Step 1 Cross-linking: Addition of formaldehyde results in the cross-linking of DNA segments to proteins and to cross-linking of proteins with each other. This leads to cross-linking of interacting DNA segments (for example cis located promoters to trans located promoters, reveals interactions like the interaction between H enhancer and odorant receptor promoters).

Step 2 Restriction digest: A restriction enzyme is added in excess to the cross-linked DNA, separating the non-cross-linked DNA from the cross-linked chromatin. The selection of the restriction enzyme in this step depends on the locus being analyzed and allows the separate analysis of different regulatory elements. Frequently cutting enzymes (4 bp) are used in studying small loci (<10–20 kb), while larger loci demand the use of larger cutters (6 bp). Restriction enzyme selection also has an impact on digestion efficiency of cross-linked DNA.

Step 3 Intramolecular Ligation:

Using very low concentrations of DNA favors the ligation of relevant DNA fragments with the corresponding junctions instead of the ligation of random fragments. There are two major types of ligation junctions that are over-represented. One is the junction that forms between neighboring DNA fragments due to incomplete digestion, which represents about 20-30% of all junctions. This number can be decreased by reducing the cross-linking stringency in the first step. The other type of junctions over-represented here is the junction that forms when one end of the fragment ligates with the other end of the same fragment, and contributes up to 30% of all junctions formed.

Step 4 Reverse Cross-links: High temperature will result in the reversal of cross-links formed in step 1. The resulting linear DNA fragment has specific restriction ends as well as a central restriction site corresponding to the site of ligation. The pool of these fragments is collectively referred to as the 3C library.

Step 5 Quantitation: Polymerase chain reaction (PCR) uses primers against the site of ligation to semi-quantitatively assess the frequencies of a restriction fragment of interest. Real-time PCR using Taqman probes (3C-qPCR) provides a more quantitative measurement of the fragment of interest. The Taqman probe and a constant primer hybridize to the restriction fragment that contains the site of contact and one test primer is designed against each neighboring restriction fragments. Together the probe and primers allow for a specific fluorescent signal to be emitted during amplification.