Genome-wide determination of mammalian replication timing by DNA content measurement

Yishai Yehuda, Britny Blumenfeld, Dan Lehmann, Itamar Simon*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge. This article describes in detail the "Copy Number Ratio of S/G1 for mapping genomic Time of Replication" method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

Original languageAmerican English
Article numbere55157
JournalJournal of Visualized Experiments
Volume2017
Issue number119
DOIs
StatePublished - 19 Jan 2017

Bibliographical note

Publisher Copyright:
© 2017 Journal of Visualized Experiments.

Keywords

  • Bioinformatics
  • FACS
  • Genetics
  • Genomics
  • Issue 119
  • Replication
  • Replication timing
  • Sequencing

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