Analysis of DNA replication by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has been shown to discriminate between unreplicated and replicated regions of the genome in interphase nuclei, based on the number of specific fluorescent signals that can be detected. By examining the replication status of hybridizing sequences in large numbers of individual cells from an asynchronously growing population, it is possible to deduce a relative order of replication of different sequences. The availability of well-mapped genomic probes and the ability to compare results from different cell lines make this a convenient approach with which to map domains of replication timing control at any chromosomal position and to relate this to various patterns of gene expression. Since there appear to be important but poorly understood correlations among replication timing, chromatin structure, and transcriptional competence in mammalian cells, this provides a valuable approach to understanding these interrelationships at the molecular level. The procedures for using FISH to examine replication timing in mammalian nuclei are described here in detail, and the advantages and limitations of the approach are discussed. Some other strategies for using high-resolution FISH on chromatin fibers to examine replication properties of specific sequences in situ are also described.