The need to combine chromosome banding with
fluorescence in situ hybridization has meant that banding
techniques using fluorescent dyes has become more
popular. Q-banding involves staining with quinacrine
which reacts specifically with certain bases. Quinacrine
intercalates into chromosomal DNA irrespective of
sequence, but fluoresces brighter in regions of AT-rich
DNA. There are a number of other molecules whose
fluorescence is influenced by the base composition of the
DNA to which they are bound. In addition to quinacrine,
other commonly used fluorochromes with a specificity for
AT-rich DNA include Hoechst 33258, DAPI (4’-6-
diamidino-2-phenylindole) and daunomycin. The fluorescence of Hoechst and DAPI is not quenched by guanine
and so they give less distinct bands than those produced by
quinacrine; however, daunomycin fluorescence is greatly
quenched by DNA with a GC content of 432%. DAPI
staining has the advantage that it is very resistant to fading
and that its excitation and emission spectra are compatible
withreportermoleculesandfilterscommonlyusedinFISH
(Figure 2).
Fluorochromes with a preference for GC-rich DNA
include chromomycin and 7-amino actinomycin D. These
dyes give an R-band-like pattern.