title: Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR).
abstract: Nascent strand capture and release (NSCR) is a method for isolation of short nascent strands to identify origins of DNA replication. The protocol provided involves isolation of total DNA, denaturation, size fractionation on a sucrose gradient, 5'-biotinylation of the appropriate size nucleic acids, binding to a streptavidin coated magnetic beads, intensive washing, and specific release of only the RNA-containing chimeric nascent strand DNA using ribonuclease I (RNase I). The method has been applied to mammalian cells derived from proliferative tissues and cell culture but could be used for any system where DNA replication is primed by a small RNA resulting in chimeric RNA-DNA molecules.
authors.affiliation: Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: Dimiter
authors.initials: D
authors.lastname: Kunnev
authors.affiliation: Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Freeland
authors.affiliation: Department of Biostatistics and Bioinformatics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: Maochun
authors.initials: M
authors.lastname: Qin
authors.affiliation: Department of Biostatistics and Bioinformatics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Pruitt
keywords: DNA replication; NSCR; SNS; origin mapping; ribonuclease I