Analysis of Replicating Yeast Chromosomes by DNA Combing

Cold Spring Harbor Protocols
David GalloGrant W Brown

Abstract

Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replication fork progression in the budding yeast Saccharomyces cerevisiae. In contrast to traditional flow cytometry, microarray, or sequencing techniques, which provide population-level data, DNA combing provides DNA replication profiles of individual molecules. DNA combing uses yeast strains that express human thymidine kinase, which facilitates the incorporation of thymidine analogs into nascent DNA. First, DNA is isolated and stretched uniformly onto silanized glass coverslips. Following immunodetection with antibodies that recognize the thymidine analog and the DNA, the DNA fibers are imaged using a fluorescence microscope. Finally, the lengths of newly replicated DNA tracks are measured and converted to base pairs, allowing calculations of the speed of the replication fork and of interorigin distances. DNA combing can be applied to monitor replication defects caused by gene mutations or by chemical agents that induce replication stress. Here, we present a methodology for studying replicating yeast chromosomes by molecular DNA combing. We begin with procedures for the preparation of silanized coverslips and for assembly of a DNA combin...Continue Reading

References

Apr 19, 2003·The EMBO Journal·Gwennaelle VersiniPhilippe Pasero
Jul 1, 2009·Methods in Molecular Biology·Etienne SchwobClaire Amato
Dec 30, 2009·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·Sandie TuduriPhilippe Pasero
May 15, 2012·Methods : a Companion to Methods in Enzymology·Julien N BiancoPhilippe Pasero
Feb 3, 2016·Cold Spring Harbor Protocols·David GalloGrant W Brown

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Citations

Jul 25, 2017·Cold Spring Harbor Protocols·Francisco Antequera, Timothy C Humphrey
Jul 1, 2018·The Journal of Biological Chemistry·Sara Rodriguez-AcebesJuan Méndez
Jul 25, 2017·Cold Spring Harbor Protocols·Divya R IyerNick Rhind
Feb 3, 2016·Cold Spring Harbor Protocols·David GalloGrant W Brown

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