Dostie Josée, Dekker Job
Program in Gene Function and Expression and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Lazare Research Building, 364 Plantation Street, Room 519, Worcester, Massachusetts 01605-4321, USA.
Nat Protoc. 2007;2(4):988-1002. doi: 10.1038/nprot.2007.116.
Genomic elements separated by large genomic distances can physically interact to mediate long-range gene regulation and other chromosomal processes. Interactions between genomic elements can be detected using the chromosome conformation capture (3C) technology. We recently developed a high-throughput adaptation of 3C, 3C-carbon copy (5C), that is used to measure networks of millions of chromatin interactions in parallel. As in 3C, cells are treated with formaldehyde to cross-link chromatin interactions. The chromatin is solubilized, digested with a restriction enzyme and ligated at low DNA concentration to promote intra-molecular ligation of cross-linked DNA fragments. Ligation products are subsequently purified to generate a 3C library. The 5C technology then employs highly multiplexed ligation-mediated amplification (LMA) to detect and amplify 3C ligation junctions. The resulting 5C library of ligated primers is analyzed using either microarray detection or ultra-high-throughput DNA sequencing. The 5C protocol described here can be completed in 13 d.
基因组距离相隔很远的基因组元件能够发生物理相互作用，从而介导远程基因调控及其他染色体过程。基因组元件之间的相互作用可通过染色体构象捕获（3C）技术来检测。我们最近开发了3C的高通量改进技术——3C碳拷贝技术（5C），该技术用于并行测量数百万个染色质相互作用网络。与3C技术一样，细胞用甲醛处理以交联染色质相互作用。染色质被溶解，用限制性内切酶消化，并在低DNA浓度下连接，以促进交联DNA片段的分子内连接。随后纯化连接产物以生成3C文库。然后，5C技术采用高度多重连接介导的扩增（LMA）来检测和扩增3C连接接头。使用微阵列检测或超高通量DNA测序分析所得的连接引物5C文库。此处描述的5C实验方案可在13天内完成。
