Methods of fluorescence in situ hybridization on extended DNA fibers (Fiber-FISH)

Fiber-fluorescence in situ hybridization (fiber-FISH) is a cytogenetic technique that can be used to visualize DNA probes on extended DNA fibers. Fiber-FISH provides a significantly higher mapping resolution than do the chromosome- or chromatin-based cytogenetic mapping techniques. Here we report a simple procedure for performing fiber-FISH. In general, we consider the overall protocol to comprise four distinct steps, each of which is critical to the success of the experiment: the isolation of nuclei from plant tissue, the extension of fibers on glass slides, the hybridization of labeled DNA probes to denatured DNA fibers, and the immunodetection of the probes. We end the protocol discussion by addressing some of the more frequently asked questions with regard to common problems encountered and the technical limitations of the procedure.