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
RESUMEN

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