The neutral/neutral and neutral/alkaline two-dimensional gel electrophoreti
c techniques are sensitive physical mapping methods that have been used suc
cessfully to identify replication initiation sites in genomes of widely var
ying complexity. We present detailed methodology for the preparation of rep
lication intermediates from mammalian cells and their analysis by both neut
ral/neutral and neutral/alkaline two-dimensional gel approaches. The method
s described allow characterization of the replication pattern of single-cop
y loci, even in mammalian cells. When applied to metazoans, initiation is f
ound to occur at multiple sites scattered throughout zones that can be as l
ong as 50 kb, with some subregions being preferred. Although these observat
ions do not rule out the possibility of genetically defined replicators, th
ey offer the alternative or additional possibility that chromosomal context
may play an important role in defining replication initiation sites in com
plex genomes. We discuss novel recombination strategies that can be used to
test for the presence of sequence elements critical for origin function if
the origin lies in the vicinity of a selectable gene. Application of this
strategy to the DHFR locus shows that loss of sequences more than 25 kb fro
m the local initiation zone can markedly affect origin activity in the zone
. (C) 1999 Academic Press.