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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.

SUBMITTER: Kunnev D

PROVIDER: S-EPMC4778562 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR).

Kunnev Dimiter D Freeland Amy A Qin Maochun M Wang Jianmin J Pruitt Steven C SC

Journal of biological methods 20151230 4

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 appl ...[more]

PMID: 26949711

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