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Chromosome conformation capture (3C) methodology was developed to study spatial organization of long genomic regions in living
cells. Briefly, chromatin is fixed with formaldehyde in vivo to cross-link interacting sites, digested with a restriction
enzyme and ligated at a low DNA concentration so that ligation between cross-linked fragments is favored over ligation between
random fragments. Ligation products are then analyzed and quantified by PCR. So far, semi-quantitative PCR methods were widely
used to estimate the ligation frequencies. However, it is often important to estimate the ligation frequencies more precisely
which is only possible by using the real-time PCR. At the same time, it is equally necessary to monitor the specificity of
PCR amplification. That is why the real-time PCR with TaqMan probes is becoming more and more popular in 3C studies. In this
chapter, we describe the general protocol for 3C analysis with the subsequent estimation of ligation frequencies by using
the real-time PCR technology with TaqMan probes. We discuss in details all steps of the experimental procedure paying special
attention to weak points and possible ways to solve the problems. A special attention is also paid to the problems in interpretation
of the results and necessary control experiments. Besides, in theory, we consider other approaches to analysis of the ligation
products used in frames of the so-called 4C and 5C methods. The recently developed chromatin immunoprecipitation (ChIP)-loop
assay representing a combination of 3C and ChIP is also discussed.
Affiliation(s): (1) CNRS UMR-8126, Université Paris-Sud 11, Institut de Cancérologie Gustave Roussy, 39, rue Camille-Desmoulins, 94805 Villejuif, France
(2) Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
(3) Vanderbilt University, Nashville, TN, USA
(2) Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
(3) Vanderbilt University, Nashville, TN, USA
Series: Methods in Molecular Biology | Volume: 567 | Pub. Date: Aug-01-2009 | Page Range: 171-188 | DOI: 10.1007/978-1-60327-414-2_12
Subject: Genetics/Genomics
Key Words: 3C - ChIP-loop assay - 4C - 5C - TaqMan probes - real-time PCR - chromatin - genome spatial organization
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