Analyzing the dynamics of DNA replication in Mammalian cells using DNA combing

Bialic, M.; Coulon, V.; Drac, M.; Gostan, T.; Schwob, E.

Methods Mol Biol

2015 / vol 1300 / pages 67-78


How cells duplicate their chromosomes is a key determinant of cell identity and genome stability. DNA replication can initiate from more than 100,000 sites distributed along mammalian chromosomes, yet a given cell uses only a subset of these origins due to inefficient origin activation and regulation by developmental or environmental cues. An impractical consequence of cell-to-cell variations in origin firing is that population-based techniques do not accurately describe how chromosomes are replicated in single cells. DNA combing is a biophysical DNA fiber stretching method which permits visualization of ongoing DNA synthesis along Mb-sized single-DNA molecules purified from cells that were previously pulse-labeled with thymidine analogues. This allows quantitative measurements of several salient features of chromosome replication dynamics, such as fork velocity, fork asymmetry, inter-origin distances, and global instant fork density. In this chapter we describe how to obtain this information from asynchronous cultures of mammalian cells.

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1940-6029 (Electronic) 1064-3745 (Linking)

IGMM team(s) involved in this publication

Animals; Mice; *DNA Replication; Biophysics/*methods; DNA/*metabolism; Embryo, Mammalian/cytology; Fibroblasts/cytology/metabolism; Image Processing, Computer-Assisted; Mammals/*metabolism; Staining and Labeling

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