Gene Mapping and PCR Applications with Flow-Sorted Chromosomes

Abstract

The bivariate analysis by flow cytometry of human chromosome suspensions stained with the two fluorochromes, Hoechst 33258 (specificity for AT-rich DNA) and Chromomycin A3 (specificity for GC-rich DNA) enables the majority of the chromosome types to be resolved and sorted (Gray et. al. 1979). In the flow cytometer, chromosomes pass sequentially through two spatially separated laser beams, the first operated using the UV lines (351–364 nm) to excite Hoechst fluorescence and the second operated at 457.9 nm to excite Chromomycin fluorescence. The fluorescence intensity of each chromosome is measured for both dyes independently and correlated on the bivariate flow karyotype where chromosome types are resolved by DNA content and base pair ratio (see Fig. 1).