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Chromosome conformation capture, or 3C, is a high-throughput molecular biology technique used to analyze the organization of chromosomes in a cell's natural state. Studying the structural properties and spatial organization of chromosomes is important for the understanding and evaluation of the regulation of gene expression, DNA replication and repair, and recombination. One example of chromosomal interactions influencing gene expression is a chromosomal region which can fold in order to bring an enhancer and associated transcription factors within close proximity of a gene, as was first shown in the beta-globin locus. Chromosome conformation capture has enabled researchers to study the influences of chromosomal activity on the aforementioned cellular mechanisms. This technology has aided the genetic and epigenetic study of chromosomes both in model organisms and in humans. Several techniques have been developed from 3C to increase the throughput of quantifying a chromosome’s interactions with other chromosomes and with proteins. All the 3C related technologies are broadly categorized into four groups. (1) 3C and ChIP version of 3C (ChIP-loop assay), (2) 4C and ChIP version of 4C (enhanced 4C), (3) 5C and 3D assays and (4) Genome conformation capture (GCC) related (Hi-C), ChIP version of GCC as 6C. The application of analyzing DNA segments by microarray and high-throughput sequencing in the 4C, 5C and Hi-C methodologies has brought the assessment of chromosome interactions to the genome-wide scale. ==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 the 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.〔Dekker J. Personal communication.〕 ''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. Quantitative 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 fragment. Together the probe and primers allow for a specific fluorescent signal to be emitted during amplification. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Chromosome conformation capture」の詳細全文を読む スポンサード リンク
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