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 column or magnetic beads, intensive washing, and specific release only the RNA containing chimeric nascent strand DNA using RNaseI. 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.