USE OF THE COMBING PROCESS FOR THE IDENTIFICATION OF DNA ORIGINS OF REPLICATION

摘要

Eukaryotic genomes are duplicated by the activation of multiple bidirectional origins of replication. The replication programs of these cells depend on the temporal and spatial organisation of replication origins throughout the genome. To investigate the replication program in a higher eukaryote, we employed a technique called molecular combing. This technique allows for a quantitative analysis of DNA replication on a genome wide basis. As a model system, Xenopus laevis sperm chromatin were differentially labelled at successive time points after the beginning of DNA synthesis. Genomic DNA was then extracted and combed on a glass surface. Direct measurements made on the labelled DNA provided a comprehensive analysis of the spatial and temporal organisation of the X. leavis early embryo replication program and revealed that the number of replication origins activated per kilobase increases throughout the period of DNA synthesis.