Mapping initiation sites of DNA replicationin vivousing polymerase chain reaction amplification of nascent strand segments

Abstract

We describe a sensitive method for mapping replication initiation sites near regions of sequenced genomic DNA in vivo. It is based on selective amplification of sets of segments in purified nascent DNA strands and subsequent determination of the lengths of these strands required to include each member of the set. We demonstrate the ability of this method to accurately map a well-defined origin, that of replicating SV40 DNA. Pulse-labeled DNA from infected CV-1 cells was size-fractionated on an alkaline sucrose gradient and newly-synthesized strands purified by immunoprecipitation using anti-BrdU antibodies. Three pairs of synthetic oligonucleotide primers were used to amplify three SV40 segments, using the polymerase chain reaction (PCR), at known distances from the origin. Lengths of the nascent DNA strands that allow amplification were determined by hybridization to probes homologous to the amplified segments and used to calculate position of the origin. Experiments with a mix of SV40 and human HeLa cell DNA demonstrate the applicability of the method to mapping origins present at the level of single-copy genomic sequences in mammalian cells.