Gene Mapping and Molecular Cytogenetics Using Flow-Sorted Chromosomes

Abstract

The bivariate analysis of human chromosome suspensions by flow cytometry (1) enables the majority of the chromosome types to be resolved and sorted. Chromosomes are stained with two DNA-specific fluorochromes, Hoechst 332 58 which has specificity for AT-rich DNA and Chromomycin A3 with specificity for GC-rich DNA. In the flow cytometer, the chromosomes are passed 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 intensity of the fluorescence emissions from the two dyes are measured independently and correlated on the bivariate flow karyotype where chromosome types are resolved by DNA content and base pair ratio (see Fig. 1). All of the chromosome types can be resolved in this way with the exception of chromosomes 9–12 which display similar DNA content and base pair ratio.