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

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

Journal of biological methods (2015-01-01)
Dimiter Kunnev, Amy Freeland, Maochun Qin, Jianmin Wang, Steven C Pruitt
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.

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Sigma-Aldrich
SeqPlex DNA Amplification Kit, For use with high throughput sequencing technologies, Whole Genome Amplification kit designed to facilitate Next Gen Sequencing.