Chromosoma (1993) 102:325-332 CHROMOSOMA 9 Springer-Verlag 1993 Modes of DAPI banding and simultaneous in situ hybridization Henry H.Q. Heng, Lap-Chee Tsui Department of Molecular and Medical Genetics, University of Toronto, and Department of Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1H8, Canada Received: 11 November 1992; in revised form: 28 January 1993/Accepted: 28 January 1993 Abstract. By controlling the degree of chromatin denatu- ration through formamide incubation, or by heat treat- ment and/or by high pH, three types of high quality 4',6- diamidino-2-phenylindole (DAPI) bands can be pro- duced sequentially on the same set of 5-bromo-2'-de- oxyuridine (BrdU)-incorporated chromosomes: first DAPI multibanding (the equivalent of Q-banding), then partial C-banding including distamycin A (DA)/DAPI banding, and finally C-banding pattern. It is assumed that the different DAPI-chromatin interactions following these treatments reflect the different chromatin structures at the chromosomal sites. Since the DAPI banding pro- tocol is compatible with in situ hybridization, the combi- nation of fluorescent in situ hybridization (FISH) with DAPI banding allows the simultaneous detection of sig- nals from the DNA probes and the identification of the chromosomal band location of the probe. We demon- strate this useful application with the localization of the cystic fibrosis and Duchenne muscular dystrophy gene probes to their appropriate bands. Introduction The chromatin-binding fluorescent dye DAPI (4'-6-dia- midino-2-phenylindole) can be used to visualize recta- phase chromosomes. The fluorescent intensity of the chromosome can be influenced by the level of DNA denaturation and by the competitive DNA binding of distamycin A (DA), thus producing chromosome-specif- ic banding patterns (Schweizer and Nagl 1976; Schweizer 1976; Lin et al. 1977). Both induced denatura- tion and competitive binding are a function of DNA structure. Investigation of the chromosome-staining behaviour with different AT-specific fluorescent dyes has led Schweizer (1983) to suggest that the positive DA/ DAPI banding may indicate different classes of hetero- Communicated by: P.B. Moens Correspondence to: H.H.Q. Heng chromatin. For example, the heterochromatin of chro- mosomes 1 and 16 shows positive fluorescence with DA/ DAPI and DAPI/actinomycin, representing AT-rich type B heterochromatin, while chromosome 9 exhibits positive DA/DAPI banding but is negative for DAPI/ actinomycin, thus representing type C heterochromatin. These AT-rich sequences in the DA/DAPI positive chro- mosomes have been compared for their AT content (Schwarzacher-Robinson etal. 1988; Moyzis etal. 1987). DNA clones of higher AT content (66%) and longer AT stretches locate to chromosomes 1 and 16 while the lower AT content clones (58%) are found on chromosome 9 (Schwarzacher-Robinson etal. 1988). Thermally stable, highly conserved repetitive DNA se- quence clones consisting of (GGAAT)n repeats have been located to the centromeric regions of human chro- mosomes as well as the adjacent heterochromatic regions of chromosomes 1, 9, 16 and Y (Grady et al. 1992). Since DAPI associates only with double-stranded DNA (Hajduk 1976; Kapuscinski and Szer 1979), these ther- mal-stable sequences are expected to resist denaturation and remain DAPI positive after other chromosomal re- gion have faded. In this study, we report the denaturation pattern along the length of the 5-bromo-2'-deoxyandine (BrdU)- incorporated chromosome by a number of techniques as visualized by DAPI staining. The patterns resemble Q-banding with modest denaturation, partial C-banding (including DA/DAPI banding) with extended treatment, and C-banding with continued treatment. Fluorescent in situ hybridization (FISH) techniques allow the visualization of specific DNA sequences on the chromosome. While FISH has greatly enhanced the success of gene mapping, it has been more difficult to attain good correlation of in situ signals with conven- tional chromosome bands. Various methodologies to al- low this correlation have been reported: G-banding be- fore or after FISH (Bhatt et al. 1988; Klever et al. 1991); generation of similar R- oi G-bands with cohybridiza- tion with Alu or L1 probes (Korenberg et al. 1988 ; Boyle et al. 1990, 1992); the combination of various fluores-