Single molecule analysis of DNA replication

We describe here a novel approach for the study of DNA replication. The app roach is based on a process called molecular combing and allows for the gen ome wide analysis of the spatial and temporal organization of replication u nits and replication origins in a sample of genomic DNA. Molecular combing is a process whereby molecules of DNA are stretched and aligned on a glass surface by the force exerted by a receding air/water interface. Since the s tretching occurs in the immediate vicinity of the meniscus, all molecules a re identically stretched in a size and sequence independent manner. The app lication of fluorescence hybridization to combed DNA results in a high reso lution (1 to 4 kb) optical mapping that is simple, controlled and reproduci ble. The ability to comb up to several hundred haploid genomes on a single coverslip allows for a statistically significant number of measurements to be made. Direct labeling of replicating DNA sequences in turn enables origi ns of DNA replication to be visualized and mapped. These features therefore make molecular combing an attractive tool for genomic studies of DNA repli cation. In the following, we discuss the application of molecular combing t o the study of DNA replication and genome stability. (C) 1999 Societe franc aise de biochimie et biologie moleculaire/Editions scientifiques et medical es Elsevier SAS.