Abstract
Background: Genetic repeat elements (interspersed or tandem repeats) have diverse functions within cells and at different phases of the cell cycle. Yet, their investigation at a genome-wide scale has been difficult ever since due to their repetitive nature. Here, we describe a method to study the DNA replication kinetics of different repeat elements in single cells throughout the S-phase of the cell cycle. Methods: Mouse major satellite, minor satellite and telomere repeat elements as well as human LINE-1 and Alu repeats are detected in Fluorescence In Situ Hybridization (FISH) assays by specific hybridization probes containing biotinylated nucleotides. We combine their visualization via fluorophore-tagged streptavidin with the detection of cellular DNA replication signals at the single-cell level. Results: All probes hybridized specifically to their intended target sequence as verified by microscopical analysis and with regard to their location on individual chromosomes. We detected DNA replication by labeling replisome components or nascent DNA synthesis by detecting incorporated thymidine analogues. The different spatial distribution of replication signals allowed us to classify the cells in different S-phase substages. We additionally measured genomic DNA content to time the progression throughout S-phase. Then, we performed colocalization analysis of the degree of signal overlap between DNA replication and DNA probe signal to determine the kinetics of DNA replication of repeat DNA in individual cells and by high-throughput/high-content microscopical analysis. Conclusions: Application of the probes mentioned above in 3-dimensionally preserved interphase nuclei with the simultaneous detection of newly synthesized DNA or components of the replication machinery allows to determine the replication kinetics of all elements during the synthesis phase of the cell cycle in human as well as embryonic and somatic mouse cells.
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