title: Analysis of Replicating Yeast Chromosomes by DNA Combing.
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 combing machine (DCM) and conclude by presenting a detailed protocol for molecular DNA combing in yeast.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Gallo
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yip
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Grant W
authors.initials: GW
authors.lastname: Brown
mesh_terms: D002455:Cell Division; D015825:Chromosomes, Fungal; D004261:DNA Replication; D008856:Microscopy, Fluorescence / Q000379:methods*; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology*