Localization of Low-Copy DNA Sequences on Mitotic Chromosomes by FISH

Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on mitotic and meiotic chromosomes and interphase nuclei. It was developed in early 1980s and since then it has contributed to numerous studies and important discoveries. Over the decades, the protocol was modified for ease of use, allowing for localizing multiple probes simultaneously and increasing its sensitivity and specificity. Despite the continuous improvements, the ability to detect short single-copy sequences of only a few kilobases or less, such as genes, remains limited. Here, we provide a detailed protocol for detection of short, single- or low-copy sequences on plant mitotic metaphase chromosomes.