DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

Abstract

By using FISH, the positions of DNA sequences can be discretely marked with a fluorescent spot. For this reason, FISH has proven useful in gene mapping, studying large-scale variation in the human genome, detecting chromosome rearrangements associated with disease, and in understanding the organization of the cell nucleus.The applications of FISH in gene mapping and cytogenetics requires a basic understanding of the organization of the interphase nucleus. These applications are constrained by the efficiency of hybridization, the spatial resolution of hybridization sites in nuclei, and the correlation (if any) between the separation of sequences in interphase chromatin and their separation on the linear DNA molecule.The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ∼0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.