Göndör Anita, Rougier Carole, Ohlsson Rolf
Department of Development and Genetics, Uppsala University, Norbyvägen 18A, S-752 36 Uppsala, Sweden.
Nat Protoc. 2008;3(2):303-13. doi: 10.1038/nprot.2007.540.
The pioneering chromosome conformation capture (3C) method provides the opportunity to study chromosomal folding in the nucleus. It is based on formaldehyde cross-linking of living cells followed by enzyme digestion, intramolecular ligation and quantitative (Q)-PCR analysis. However, 3C requires prior knowledge of the bait and interacting sequence (termed interactor) rendering it less useful for genome-wide studies. As several recent reports document, this limitation has been overcome by exploiting a circular intermediate in a variant of the 3C method, termed 4C (for circular 3C). The strategic positioning of primers within the bait enables the identification of unknown interacting sequences, which form part of the circular DNA. Here, we describe a protocol for our 4C method, which produces a high-resolution interaction map potentially suitable for the analysis of cis-regulatory elements and for comparison with chromatin marks obtained by chromatin immunoprecipitation (ChIP) on chip at the sites of interaction. Following optimization of enzyme digestions and amplification conditions, the protocol can be completed in 2-3 weeks.
开创性的染色体构象捕获（3C）方法为研究细胞核中的染色体折叠提供了契机。它基于活细胞的甲醛交联，随后进行酶消化、分子内连接和定量（Q）-PCR分析。然而，3C需要事先了解诱饵和相互作用序列（称为相互作用体），这使得它在全基因组研究中的用途较小。正如最近的几份报告所记载的，通过利用3C方法的一个变体（称为4C，即环状3C）中的环状中间体，这一局限性已被克服。引物在诱饵内的策略性定位能够识别未知的相互作用序列，这些序列构成环状DNA的一部分。在此，我们描述了我们的4C方法的实验方案，该方案可产生高分辨率的相互作用图谱，可能适用于顺式调控元件的分析，并可与在相互作用位点通过芯片染色质免疫沉淀（ChIP）获得的染色质标记进行比较。在优化酶消化和扩增条件后，该方案可在2至3周内完成。
