Chromosome measurement and sorting by flow systems.

Abstract

A flow microfluorometer was used to measure metaphase chromosomes in suspension at rates up to 100,000 per min. Chromosomes from cells of the Chinese hamster M3-1 cell line were isolated, stained for DNA with the fluorescent dye ethidium bromide, and analyzed for DNA content. Nine distinct peaks were resolved that correspond well with independent chromosomal DNA measurements made with a high-resolution scanning cytophotometer. Chromosomes were sorted from each peak by an electronic cell sorter. Visual examination of each fraction indicated the purity of the sorted chromosomes. This novel technology allows separation of purified populations of individual chromosomes suitable for biochemical and biological characterizations. Metaphase chromosomes can be described by several properties, including length, area, DNA content, and sedimentation velocity (1, 2). Of these, DNA content is the most fundamental chromosomal descriptor and the only one insensitive to shape and degree of compaction. Heretofore, chromosomal DNA content has been measured cytophotometrically in conventional slide preparations (2-4); the method is complex and time-consuming, and cannot be used to prepare fractions of purified chromosomes. Bulk techniques such as centrifugation can sort the chromosomes, but only into gross fractions of low purity (5). The advent of flow microfluorometry and flow sorters allows application of precise DNA measurement, rapid processing, and individual sorting to metaphase chromosomes. We report here the first such application. Individual chromosomes of a clonal derivative (650A) from the M3-1 Chinese hamster cell line were isolated, stained for DNA with ethidium bromide, and analyzed by the flow system. A distribution of DNA for the chromosomes in suspension was obtained that showed nine distinct peaks. The chromosomes corresponding to each peak were separated for visual scoring by an electronic cell sorter. The relative DNA content (peak mean), chromosomal frequency (peak area), and chromosome type (peak content as determined visually) were compared with measurements of similar parameters made with a high-resolution scanning cytophotometer on chromosomes from metaphase spreads of cells from the same culture. The agreement was excellent. Chromosome spreads of cells from the same culture were also prepared for karyotyping and analysis by CYDAC (computer-oriented image analysis microscope system) according to procedures described elsewhere (2).Chromosome Staining. For flow system analysis, the chromosomes, still suspended in the Pipes solution, were stained for DNA by adding an equal volume of 0.004% ethidium bromide in Pipes buffer. The chromosomes were uniformly stained within 10 min; they remained in the stain solution throughout flow system analysis. Chromosomal fluorescence was stable from 0.1 hr to 2 days after staining.Other DNA dyes were used also. Propidium iodide, substituted for ethidium bromide, gave almost identical results. Chromosomes prepared according to the procedure of Burki et al. (5) and sta...

The DNA‐based human karyotype

“…Flow measurements of chromosome DNA content agree closely with our measurements of chromosome DNA content when the chromosomes are stained for flow with dyes, such as propidium iodide or ethidium bromide, that show no base specificity (6, 18,19 19 show less. Chromosomes 4 and 5, which have nearly identical DNA contents (Table 3), show an 8% difference in Hoechst 33258 fluorescence.…”

Orientation measurements of microsphere doublets and metaphase chromosomes in flow

“…Cell culture procedures for Chinese hamster M3-1 cells were identical with those described elsewhere (2,4,14). Chromosomes in this study were isolated according to the procedure described by Aten (1).…”

“…Orientation studies of Chinese hamster M3-1 metaphase Chromosomes showed that over 95% of the larger chrumosomes (1)(2)(3)(4)(5) and over 65% of the smaller chromosomes (9)(10)(11) were oriented near the entrance of the capillary tube.…”

An appraisal of the application of recombinant DNA techniques to chromosome defects

“…Probes may be isolated from total genomic libraries (Maniatis et al, 1978;Dahl et al, 1981), but as the X chromosome only comprises 5.4% of the human genome (Gray et al, 1975) a method of enrichment for chromosome-specific sequences is desirable. Three main approaches have been employed to obtain chromosome-specific DNA.…”