We have developed epifluorescence filter sets and computer software for the detection and discrimination of 27 different DNA probes hybridized simultaneously. For karyotype analysis, a pool of human chromosome painting probes, each labelled with a different fluor combination, was hybridized to metaphase chromosomes prepared from normal cells, clinical specimens, and neoplastic cell lines. Both simple and complex chromosomal rearrangements could be detected rapidly and unequivocally; many of the more complex chromosomal abnormalities could not be delineated by conventional cytogenetic banding techniques. Our data suggest that multiplex-fluorescence in situ hybridization (M-FISH) could have wide clinical utility and complement standard cytogenetics, particularly for the characterization of complex karyotypes.

Fluorescence in situ hybridization has been used to demonstrate the differential distribution of interspersed repetitive elements in the genome ofMus musculus domesticus. Hybridization with a mouse long interspersed element sequence results in a sharp, highly reproducible banding pattern on metaphase chromosomes, which is quite similar to Giemsa banding for all chromosomes except 7 and X. The families of short interspersed elements, B1 and B2, preferentially cluster in the R, or reverse, bands. There is no evidence of any interspersed repeat present in the centromeric heterochromatic regions. Both the long interspersed element and B2 probes give banding patterns suitable for karyotype analysis. Simultaneous hybridization of the biotinylated long interspersed element probe and a digoxigenin-labeled cosmid to metaphase spreads allows rapid localization of a probe of interest to a particular cytogenetic band on a chromosome.

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Proteins encoded by homeobox containing genes are sequence-specific DNA binding proteins implicated in the control of gene expression in both developing and adult tissues. Two recently characterized human homeobox genes, HB9 and HB24, are highly expressed in CD34-positive marrow cells but not in CD34-depleted marrow cells. Their expression is readily down-regulated during the differentiation of hematopoietic progenitors to specific cell lineages. In this study, genomic DNA fragments isolated with HB9 (3 kb) and HB24 (6 kb) cDNAs were used to map their chromosomal location by fluorescence in situ hybridization. Both HB9 and HB24 DNA probes gave specific hybridization signals on chromosome 1. The hybridization loci were identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by cohybridization in situ with an Alu probe (R-like banding) and by DAPI staining (G-like). The results demonstrate that the loci of the HB24 and HB9 genes are within bands 1q41-q42.1. A cohybridization experiment utilizing both probes with two-color fluorescence imaging could not resolve separate loci for the two genes.