Analyzing the dynamics of DNA replication in Mammalian cells using DNA combing.
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.
Institute of Molecular Genetics, CNRS UMR5535 & Université Montpellier, 1919 route de Mende, Montpellier, 34293, France.
Marta
M
Bialic
Vincent
V
Coulon
Marjorie
M
Drac
Thierry
T
Gostan
Etienne
E
Schwob
D000818:Animals; D001703:Biophysics / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008322:Mammals / Q000378:metabolism*; D051379:Mice; D013194:Staining and Labeling
D004247:DNA