![]() Interacting fragments are then ligated together and purified, creating a genomic library of chimeric DNA molecules ( Figure 1C). Once interacting loci are crosslinked, chromatin is solubilized and fragmented, usually using a restriction enzyme ( Figure 1B). The 3C technique and its derivatives measure the population-averaged frequency at which two DNA fragments physically associate in three-dimensional (3D) space, based on the propensity for those two locations to become formaldehyde-crosslinked together ( Figure 1A). In molecular assays based on Chromosome Conformation Capture (3C) the genomic sequence itself is the output, completely connecting chromosome structure and the genomic sequence. ![]() Using sequence specific probes in Fluorescent In Situ Hybridization (FISH) assays allows one to connect nuclear architecture and DNA sequence, but these methods are currently still limited in throughput, allowing analysis of only a few loci simultaneously. The major disadvantage of electron microscopy is its lack of connectivity to the genomic sequence, in that the incredible fine resolution architecture cannot be assigned to a specific location in the genome. Electron microscopy is used to analyze the architecture of the nucleus in nm scale detail, while fluorescent (light) microscopy provides information on the shape and distribution of specific chromosomes and chromosomal loci as well as the co-association of specific loci with sub-nuclear compartments with a resolution of 50-100 nm. These techniques can be broadly classified into microscopic and molecular assays. Many techniques are now available to observe the spatial organization of chromatin. It will also enhance the understanding of the biophysics of chromatin, and further enable the investigation of pathologies related to genome instability or nuclear morphology. The study of the packaging and organization of chromatin in the nucleus will shed light on the spatial aspects of gene regulation, chromosome morphogenesis, and genome stability and transmission. The organization of the chromatin in the nucleus is extremely relevant to biological function at the gene level as well as the global nuclear level. The long DNA strands of every cell’s genome are packaged into chromatin in a very confined nuclear volume. This spatial context will provide a new perspective to studies of chromatin and its role in genome regulation in normal conditions and in disease. A massively parallel survey of chromatin interaction provides the previously missing dimension of spatial context to other genomic studies. This advance gives Hi-C the power to both explore the chromatin biophysics as well as the implications of chromatin structure in the biological functions of the nucleus. The compatibility of Hi-C with next generation sequencing platforms makes it possible to detect chromatin interactions on an unprecedented scale. In Hi-C, a biotin-labeled nucleotide is incorporated at the ligation junction, making it possible to enrich for chimeric DNA ligation junctions when modifying the DNA molecules for deep sequencing. The ligation products contain the information of not only where they originated from in the genomic sequence but also where they reside, physically, in the 3D organization of the genome. ![]() This method is based on Chromosome Conformation Capture, in that chromatin is crosslinked with formaldehyde, then digested, and re-ligated in such a way that only DNA fragments that are covalently linked together form ligation products. We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. ![]()
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