Got your daily dose of vitamin C yet? Well in case you have, here are five more. 5C is useful for examining interactions with particular loci of interest in detail. 5C uses special primers that anneal across the ligated junction of the DNA fragments. The tails of the primers contain a universal sequence which allows amplification of all ligation products. This carbon copy library is characterized using either microarray or sequencing.
The major advantage of 5C over 4C and 3C is its large scale multiplexing which allows the creation of a larger scale interaction matrix, giving more accurate 3D reconstructions (Dekker et al., 2013). However, 5C is not a replacement for 4C. First, not all sites permit the design of 5C primers, meaning that 4C can have higher resolution. Also, 5C require each primer needed to be designed, meaning that it is not feasible to interrogate the entire genome, as millions of primers would be needed. 5C was introduced in 2007 (Dostie and Dekker, 2007). Since, it has been used to understand many processes including the role of ncRNAs in Hox gene regulation (Wang et al., 2011). 5C was recently combined with Hi-C to uncover the organization of chromosomes during mitosis (Naumova et al., 2013)
5C Additional Reading
This paper applies 5C to understand large-scale looping interactions in the human genome. The authors also integrate data from the ENCODE project to show how epigenetic marks influence chromosome looping.
The organization of bacterial chromosomes has been assumed to be random. Recent 5C data are challenging this assumption. In this review, the results of 5C experiments in bacteria are presented and show that the chromosome is actually highly organized.
- Dekker, J., Marti-Renom, M.A., and Mirny, L.A. (2013). Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data. Nat. Rev. Genet. 14, 390-403.
- Dostie, J., and Dekker, J. (2007). Mapping networks of physical interactions between genomic elements using 5C technology. Nat. Protoc. 2, 988-1002.
- Naumova, N., Imakaev, M., Fudenberg, G., Zhan, Y., Lajoie, B.R., Mirny, L.A., and Dekker, J. (2013). Organization of the mitotic chromosome. Science 342, 948-953.
- Wang, K.C., Yang, Y.W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B.R., Protacio, A., Flynn, R.A., Gupta, R.A., et al. (2011). A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120-124.