Modes of DAPI banding and simultaneous in situ hybridization

Abstract

By controlling the degree of chromatin denaturation through formamide incubation, or by heat treatment and/or by high pH, three types of high quality 4',6-diamidino-2-phenylindole (DAPI) bands can be produced sequentially on the same set of 5-bromo-2'-deoxyuridine (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 protocol is compatible with in situ hybridization, the combination of fluorescent in situ hybridization (FISH) with DAPI banding allows the simultaneous detection of signals from the DNA probes and the identification of the chromosomal band location of the probe. We demonstrate this useful application with the localization of the cystic fibrosis and Duchenne muscular dystrophy gene probes to their appropriate bands.