Chromosome Conformation Capture - Circularized Chromosome Conformation Capture (4C)

Circularized Chromosome Conformation Capture (4C)

The 4C strategy has a significant advantage over 3C in that only the sequence of one of the site of interest needs to be known. The fragment, known as the “bait”, contains the site that associates with other chromosomal regions. The 4C procedure follows the same steps as in 3C, except additional processing is needed before quantification of the fragments of interest.

Steps 1-4: See procedure listed in 3C. 6-bp-cutters are preferred in step 2.

Step 5a Second Restriction Digest: After the reversal of the cross-linked DNA, the restriction fragments are subjected to another round of restriction digest, this time with a frequent cutter that will result in smaller fragments with restriction ends that differ from the central restriction site (ligation junction).

Step 5b Self-circularization: Self-circularization of the DNA fragments is more favored now that they are not bound to other proteins or fragments. Intramolecular ligation occurs to induce the formation of the circular fragments. The pool of circular fragments becomes the 4C library.

Step 5c Inverse PCR and Quantitation: Primers are designed against the outer restriction sites of the “bait” sequence, which result in the amplification of the small unknown captured fragment. Large-scale sequencing can be used to sequence the 4C library. Custom microarrays can also be made using probes designed against the adjacent upstream and downstream regions of all genomic sites of the restriction enzyme used in step 2.