Sullivan Beth A
Department of Molecular Genetics and Microbiology, Institute for Genome Sciences & Policy, Duke University, Durham, NC, USA.
Methods Mol Biol. 2010;659:99-115. doi: 10.1007/978-1-60761-789-1_7.
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.
免疫荧光（IF）和荧光原位杂交（FISH）是用于研究中期染色体和间期细胞核的结构与组织的传统方法。使用这些技术，可以在千碱基或兆碱基分辨率下评估整条染色体区域、染色质亚结构域和特定DNA序列的位置。通过在伸展的染色质纤维上联合使用IF-FISH，甚至可以实现对蛋白质和DNA空间关系的更高分辨率观察。这种光学作图方法是一种强大的系统，可用于沿着释放的染色质纤维定位分子探针，并可视化小（<20 kb）或大（20 - 5000 kb）的染色体区域。染色质纤维分析可以填补经典染色体研究和分子分析（如在单个或多个核小体水平评估染色质组织的染色质免疫沉淀（ChIP））之间的分辨率差距。在本章中，将介绍染色质纤维IF-FISH的概念和技术方面，以及成功应用的实例。
