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.
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
1940-6029 (Electronic) 1064-3745 (Linking)
IGMM team(s) involved in this publication
DNA Replication, Genome Instability & Cell Identity
Animals; Mice; *DNA Replication; Biophysics/*methods; DNA/*metabolism; Embryo, Mammalian/cytology; Fibroblasts/cytology/metabolism; Image Processing, Computer-Assisted; Mammals/*metabolism; Staining and Labeling