Optical mapping of protein-DNA complexes on chromatin fibers.
Immunofluorescence (IF) and Fluorescence in situ Hybridization (FISH) are conventional methods used to study the structure and organization of metaphase chromosomes and interphase nuclei. Using these techniques, the locations of whole chromosome territories, chromatin subdomains, and specific DNA sequences can be evaluated at kilobase or megabase resolution. Even higher resolution of the spatial relationships of proteins and DNA can be achieved using combined IF-FISH on extended chromatin fibers. This method of optical mapping is a powerful system for localizing molecular probes along released chromatin fibers and visualizing small (<20 kb) or large (20-5,000 kb) chromosomal domains. Chromatin fiber analysis can fill the gaps in resolution between classical chromosome studies and molecular analyses, such as chromatin immunoprecipitation (ChIP) that evaluates chromatin organization at the level of single or multiple nucleosomes. In this chapter, the conceptual and technical aspects of chromatin fiber IF-FISH are presented, along with examples of successful applications.
Sullivan
Beth A
BA
Department of Molecular Genetics and Microbiology, Institute for Genome Sciences & Policy, Duke University, Durham, NC, USA. beth.sullivan@duke.edu
D000818:Animals; D002460:Cell Line; D002498:Centrifugation; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde / Q000378:metabolism; D006801:Humans; D015336:Molecular Probe Techniques; D055620:Optical Phenomena; D011506:Proteins / Q000378:metabolism*; D016707:Tissue Fixation
D002843:Chromatin; D011506:Proteins; D005557:Formaldehyde; D004247:DNA