Dec 1, 1973

Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes

Proceedings of the National Academy of Sciences of the United States of America
S A Latt

Abstract

Fluorescence of the dye 33258 Hoechst bound to chromosomes is partially quenched by the introduction of BrdU into chromosomal DNA. This finding has allowed microfluorometric detection of DNA synthesis in human metaphase chromosomes. Incorporation, shortly before cell harvest, of a pulse of thymidine into chromosomes otherwise substituted with BrdU results in sharply defined foci of bright 33258 Hoechst fluorescence, corresponding to regions of late DNA replication. The latereplicating X chromosome can thereby be clearly identified, and the time course of appearance of its fluorescent bands can be compared with that of its earlier replicating homologue. Growth of cells in medium containing BrdU for two generations allows fluorometric documentation of the semiconservative distribution of newly replicated DNA between sister chromatids, and regions of sister chromated exchange are demarcated. This approach should constitute, in many instances, a convenient, high-resolution fluorometric alternative to autoradiography.

Mentioned in this Paper

Study, Methodological
Thymidine
Mitotic Metaphase
Sex Chromosomes
Immunofluorescence Microscopy
Metaphase
Fluorescence Spectroscopy
Fluorescent stain
X Chromosome
Leukocytes

About this Paper

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