| Popis: |
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. |
