Protocol abstract
Nature Protocols 3, - 303 - 313 (2008)
Published online: 7 February 2008 | doi:10.1038/nprot.2007.540
Subject Categories: Genetic analysis | Genomics and proteomics | Isolation, purification and separation | Nucleic acid based molecular biology
High-resolution circular chromosome conformation capture assay
Anita Göndör1, Carole Rougier1 & Rolf Ohlsson1
Abstract
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.
- Department of Development and Genetics, Uppsala University, Norbyvägen 18A, S-752 36 Uppsala, Sweden.
Correspondence to: Anita Göndör1 e-mail: anita.gondor@ebc.uu.se
nature-products
A-Z product listing
- 10
T4 DNA ligase reaction buffer(Sigma-Aldrich) - 12
l complete protease inhibitor(Roche) - 5 Phusion HF Buffer(Finnzymes)
- Agilent 2100 bioanalyzer(Agilent Technologies)
- Biofuge Pico(Eppendorf)
- Cell scraper, 25 cm(Corning)
