An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization.

Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes c...