5C
Chromatin Conformation Capture Carbon Copy
5C (Dostie et al., 2007) allows concurrent determination of interactions among multiple sequences and is a high-throughput version of 3C (Sajan et al., 2012). In this method, DNA-protein complexes are crosslinked using formaldehyde. The sample is fragmented and the DNA ligated and digested with restriction enzymes. The resulting DNA fragments are amplified using ligation-mediated PCR and sequenced. Deep sequencing provides base-pair resolution of the ligated fragments.
Advantages:
- Different from 4C, 5C provides a matrix of interaction frequencies for many pairs of sites (de Wit et al., 2012)
- Can be used to reconstruct the (average) 3D conformation of larger genomic regions
Disadvantages:
- Requires a priori information of the regulatory sites (Sati et al., 2016)
- Detection may not necessarily mean an interaction, resulting from random chromosomal collisions
- Cannot scale to genome-wide studies that would require a large amount of primers
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;
Reuter J. A., Spacek D. V. and Snyder M. P. High-throughput sequencing technologies. Mol Cell. 2015;58:586-597
References:
Smith E. M., Lajoie B. R., Jain G. and Dekker J. Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus. Am J Hum Genet. 2016;98:185-201