HIGH-RESOLUTION DNA FIBER-FISH ON YEAST ARTIFICIAL CHROMOSOMES - DIRECT VISUALIZATION OF DNA-REPLICATION

Fluorescent in situ hybridization (FISH) is a powerful, direct and sensitive technique with a wide resolution range that enables the simultaneous study of multiple targets, labelled in different colours. Spreading techniques, denoted here as ‘Fiber-FISH’, increase FISH-resolution to the DMA fiber, using decondensed nuclear DMA as hybridization target1–5. FISH could be a powerful analytical tool for thorough physical examination of yeast artificial chromosomes (YACs) which are often chimaeric or contain internal deletions. However, with one exception restricted to meiotic yeast chromosomes6, FISH has not been used successfully on yeast/YAC DMA. We have developed a fast and simple method that can be applied routinely for compositional and structural analysis of cosmid and YAC DMA in yeast. It enables precise localization and ordering of clones, resolves overlaps and distances and gives a detailed picture of the integrity and colinearity of both probe and target. The combination of high resolution, signal abundance and short yeast cell cycle allows direct visualization of replicating DMA fibers. In a 400 kb region of the human dystrophin gene, we identified two replication origins, demonstrating that human DMA cloned in yeast is capable of initiating its own replication. © 1995 Nature Publishing Group.