MAPPING REPLICATION ORIGINS BY QUANTIFYING RELATIVE ABUNDANCE OF NASCENT DNA STRANDS USING COMPETITIVE POLYMERASE CHAIN-REACTION

A procedure was developed for mapping origins of DNA replication in ma mmalian cell chromosomes based on determining the relative abundance o f nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from rec ently activated origins and the quantification, within this sample, of the relative abundances dances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin map ping proced ure (i) allows analysis of single-copy genomic regions, (ii) can be pe rformed on cultured and primary cells in the absence of any chemical t reatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of the cis- and trans-acting elements re quired for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an similar to 500-bp segment in the human lamin B2 gene domain and wi thin an similar to 8OO-bp segment in the hamster dihydrofotate reducta se gene locus. (C) 1997 Academic Press.