High resolution DNA Fiber–fish on yeast artificial chromosomes: direct visualization of DNA replication

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