Non-denaturing fluorescence in situ hybridization to find replication origins in a specific genome region on the DNA fiber.

Fluorescence in situ hybridization (FISH) is a useful method of determining the replication timing of specific genomic loci in mammals and of delineating replicon structures on DNA fibers in combination with in vivo replication labeling. In the case of simultaneous detection of a FISH probe and replicated forks, however, the DNA fibers are damaged by the DNA denaturation step for FISH detection, and the resulting fragmented fluorescence signals prevent analysis at high resolution. Here we found that hybridization of the probe to the genomic DNA was possible even under non-denaturing condition, but only at the time its genomic region replicated. Using the method designated non-denaturing FISH, we determined the replication timing of a specific BAC clone and the standard clones, and found that at least one replication origin exists within the genomic region covered by its BAC clone as an example.