Characterization of cosmids and telomeres in cytogenetic preparations by 3D confocal fluorescence.

OBJECTIVE: To analyze, with fluorescent probes, by three-dimensional (3D) emission patterns, fluorescence in situ hybridization (FISH) preparations (cosmids, telomeres) and to perform factor analysis of medical image sequences (FAMIS); to use FISH to track relevant DNA sequences in cell nuclei during interphase and in mitotic chromosomes; and to use cytogenetic techniques, resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets. STUDY DESIGN: The study design entailed labeling targets by probes (sequences labeled by fluorescein isothiocyanate) in nuclei and/or chromosomes stained by propidium iodide. Visualization of targets was improved when 3D sequences of images obtained on a single photomultiplier detector of the confocal microscope by z stepping were investigated by FAMIS. RESULTS: Factors and factor images showed that targets could be detected and differentiated in focal planes inside nuclei or chromosomes. CONCLUSION: It is possible to localize cosmids in cell nuclei at interphase and telomeres in mitotic chromosomes by means of 3D sequences of images.

Mechanically stretched chromosomes as targets for high-resolution FISH mapping.

When used with metaphase chromosomes, fluorescence in situ hybridization (FISH) makes it possible to localize probes to individual chromosome bands and to establish the order of probes separated by > or = 2-3 Mb in dual-color hybridizations. We evaluated the use of mechanically stretched chromosomes as hybridization targets for increased mapping resolution. Mapping resolution was tested by pair-wise hybridizations with probes from the 1p32-p33 region, spanning distances from 20 to approximately 1500 kb. Probes separated by > or = 170 kb could be ordered relative to one another and to the centromere-telomere axis of the chromosome. The advantages of the technique are the simple procedure for preparing the slides, the straightforward interpretation of the results, and the ability to score the predominant order from < 10 stretched chromosomes. However, because of the variability of stretching from one sample to another, the calculation of actual physical distances between probes is not possible. To illustrate the utility of this method, we showed that the gene for receptor tyrosine kinase TIE lies centromeric to COL9A2, RLF, and L-MYC genes at 1p32. The use of mechanically stretched chromosomes provides < or = 10-fold increased mapping resolution as compared with conventional metaphase FISH. Thus, the technique effectively bridges the gap between metaphase mapping and ultra-high-resolution mapping (1-300 kb) techniques, such as the DNA fiber FISH.