Separation and analysis of human chromosomes by combined velocity sedimentation and flow sorting applying single- and dual-laser flow cytometry.

Abstract

Human chromosomes were separated on basis of size by velocity sedimentation at 52g in a specially designed sedimentation chamber. The chamber has been constructed in such a way that large numbers of chromosomes can be fractionated on a sucrose gradient while wall sedimentation, streaming, and swirling movements of the gradient during centrifugation are eliminated. Flow deflectors in the chamber allow undisturbed introduction and fractionation of the density gradient. The different chromosomal fractions obtained are highly enriched for the various human chromosomes. Individual chromosomes were subsequently sorted to purity by fluorescence activated flow sorting using a FACS IV flow sorter equipped with a 4-W argon-ion laser. Following this procedure, the sorting rate for specific chromosomes can be speeded up by a factor of 5-10 when the preenriched chromosomal fractions are used as starting material. A high chromosome resolution could be obtained by a few simple modifications of the FACS IV. By applying fluorochromes with different DNA-base specificity the sorting possibilities of individual human chromosomes can be improved. In addition, the chromosomal fractions were analysed by dual-laser flow cytometry after staining the chromosomes with Hoechst 33258 and chromomycin A3. In this way the enrichment of virtually all individual human chromosomes in the different chromosomal fractions can be visualized.