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Background: Genetic repeat elements (interspersed or tandem repeats) have diverse functions within cells and at different phases of the cell cycle. However, their investigation at a genome-wide scale is challenging 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 were detected in fluorescence in situ hybridization (FISH) assays using specific hybridization probes containing biotinylated, digoxigenin-conjugated or fluorescently-labeled nucleotides. We combined their visualization with the detection of cellular DNA replication signals at the single cell level. Results: Specific hybridization of all probes to their intended target sequence and their location on individual chromosomes were verified by microscopic analysis. DNA replication OBM Genetics 2019; 3(1), doi:10.21926/obm.genet.1901062 Page 2/31 was detected by labeling replisome components or nascent DNA synthesis by detecting incorporated thymidine analogs. The different spatial distribution of replication signals allowed us to classify the cells in different S-phase substages. We also measured changes in genomic DNA content during the progression through S-phase. Using high-throughput/highcontent microscopic analysis, we then performed colocalization analysis of the degree of overlap between DNA replication and DNA probe signals to determine the kinetics of DNA replication of repeat elements in individual cells. Conclusions: Application of these probes in 3-dimensionally preserved interphase nuclei with the simultaneous detection of newly synthesized DNA or components of the replication machinery allows determination of the replication kinetics of all elements during the synthesis phase of the cell cycle in human cells as well as in embryonic and somatic mouse cells.

obm genetics

Repli-FISH (Fluorescence &lt;em&gt;in Situ&lt;/em&gt; Hybridization): Application of 3D-(Immuno)-FISH for the Study of DNA Replication Timing of Genetic Repeat Elements