Flow cytometric analysis and sorting of chromosomes
For analysis by flow cytometry, metaphase chromosomes are isolated from mitotic cells, and then stained while in suspension with one or more usually two, DNA-specific fluorescent dyes. The chromosomes are then processed one at a time through a flow cytometer or sorter where their dye content is measured at high speed and with high precision. In the work described here, dual staining is used since bivariate analysis affords better discrimination among chromosomal types: two dyes commonly used are Hoechst 33258, which binds preferentially to AT-rich DNA, and chromomycin A3, which bind preferentially to GC-rich DNA. For bivariate flow studies the chromosomes pass sequentially through 2 laser beams; the wavelength of one laser beam is adjusted to match the absorption maximum of one dye, and the wavelength of the other laser beam is adjusted to match the absorption maximum of the other dye. Thus each chromosome emits two fluorescent signals which give information on its DNA content and base composition. In typical measurements lasting a few minutes, about 10/sup 5/ chromosomes are analyzed. The bivariate fluorescence distribution accumulated by the electric processing units contains quantitative information about the chromosome population in the form of peaks produced by one or more chromosomal types. This distribution is called a flow karyotype, in analogy to the standard karyotype obtained by the cytogeneticist via banding techniques. In a flow sorter, the two fluorescent signals generated by each chromosome are used to trigger sorting of specific chromosomal types. This report reviews the current work in this area at Lawrence Livermore National Laboratory, including the work on the detection of chromosomal aberrations and on chromosome purification by sorting. 28 refs., 9 figs., 1 tab.
- Research Organization:
- Lawrence Livermore National Lab., CA (USA)
- DOE Contract Number:
- W-7405-ENG-48
- OSTI ID:
- 6476956
- Report Number(s):
- UCRL-96858; CONF-8703142-2; ON: DE87012241
- Country of Publication:
- United States
- Language:
- English
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