Chromosome conformation capture.
Chromosome conformation capture.
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DOI：
10.1101/pdb.prot5137
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发表时间：
2009-02-01
影响因子：
--
通讯作者：
Fraser, Peter
中科院分区：
文献类型：
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作者：
Cope, Nathan F;Fraser, Peter
染色体构象捕获(3C)是一种用于检测固定细胞中染色体DNA的空间组织的技术。可以对细胞核中空间邻近或参与物理相互作用(如基因和调控元件之间的DNA序列)的DNA序列进行定量评估，以提供一种潜在地反映它们关联频率和/或它们邻近程度的度量。3C可用于研究同一染色体上的DNA序列之间的远程相互作用(染色体内)或不同染色体之间的远程相互作用(染色体间)。简而言之，用甲醛固定邻近的染色质片段，然后用限制性内切酶消化，核裂解，稀释交联的复合体，以及分子内连接。随后去除交联键，提纯DNA。然后，使用针对一对正在研究的DNA序列的特异引物，利用聚合酶链式反应(PCR)扩增包含新的连接连接的DNA片段。在这里，我们描述了一个详细的3C协议和实验对照，可以应用于研究任何两个基因组区域的核并列，顺式或反式。
Chromosome conformation capture (3C) is a technique used to detect the spatial organization of chromosomal DNA in fixed cells. DNA sequences in spatial proximity in the nucleus or engaged in physical interactions (such as those between genes and regulatory elements) can be assessed quantitatively to provide a measure that potentially reflects their frequency of association and/or their proximity. 3C can be used to study long-range interactions between DNA sequences on the same chromosome (intrachromosomal) or between different chromosomes (interchromosomal). Briefly, chromatin fragments in proximity are fixed with formaldehyde, followed by digestion with restriction enzymes, nuclear lysis, dilution of the cross-linked complexes, and intramolecular ligation. The crosslinks are subsequently removed and the DNA is purified. Polymerase chain reaction (PCR) is then used to amplify DNA fragments containing novel ligation junctions using primers specific for a pair of DNA sequences under investigation. Here we describe a detailed 3C protocol and experimental controls that can be applied to investigate the nuclear juxtaposition of any two genomic regions, in cis or trans.