title: Molecular Combing of Single DNA Molecules on the 10 Megabase Scale.
abstract: DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200-500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions.
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Atanas
authors.initials: A
authors.lastname: Kaykov
authors.affiliation: Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Taillefumier
authors.affiliation: Genomic Vision, 80-84 rue des Meuniers, 92220 Bagneux, France.
authors.forename: Aaron
authors.initials: A
authors.lastname: Bensimon
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.|The Francis Crick Institute, Lincoln's Inn Fields Laboratories, London WC2A 3LY, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Nurse
mesh_terms: D002460:Cell Line; D015825:Chromosomes, Fungal / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D023281:Genomics / Q000379:methods; D006801:Humans; D018741:Replication Origin / Q000235:genetics; D016196:S Phase / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*
chemical_list: D004271:DNA, Fungal