Journal
METHODS
Volume 57, Issue 2, Pages 140-148Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2012.05.006
Keywords
DNA foci; Replication factories; Microscopy; Live cell imaging; Indirect immuno-fluorescence; S phase timing programme
Funding
- BBSRC
- CONACyT (National Council for Science and Technology, Mexico)
- Biotechnology and Biological Sciences Research Council [BBS/B/06091] Funding Source: researchfish
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The precise regulation of DNA replication is fundamental to the preservation of intact genomes during cell proliferation. Our understanding of this process has been based traditionally on a combination of techniques including biochemistry, molecular biology and cell biology. In this report we describe how the analysis of the S phase in mammalian cells using classical cell biology techniques has contributed to our understanding of the replication process. We describe traditional and state-of-the-art protocols for imaging sites of DNA synthesis in nuclei and the organisation of active replicons along DNA, as visualised on individual DNA fibres. We evaluate how the different approaches inform our understanding of the replication process, placing particular emphasis on ways in which the higher order chromatin structures and the spatial architecture of replication sites contribute to the orderly activation of defined regions of the genome at precise times of S phase. Crown Copyright (c) 2012 Published by Elsevier Inc. All rights reserved.
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