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Updated: Sep 8, 2025
Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization
Published on: December 10, 2012
Protocol for genome-wide DNA replication timing analysis using click chemistry-based biotinylation.
Deniz Gökbuget1, Kayla Lenshoek1, Robert Blelloch1
1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA; Department of Urology, University of California, San Francisco, San Francisco, CA, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
We developed BioRepli-seq, a new method to map genome-wide DNA replication timing (RT) in proliferating cells. This technique uses biotinylation and next-generation sequencing for precise RT profiling.
Area of Science:
- Genomics
- Molecular Biology
- Cell Biology
Background:
- DNA replication timing (RT) defines the cell-type-specific order of genome duplication during S phase.
- Understanding RT is crucial for deciphering genome regulation and cellular processes.
Purpose of the Study:
- To present BioRepli-seq, a novel biotinylation-based method for genome-wide RT determination.
- To provide a detailed protocol for applying this technique to any proliferating cell type.
Main Methods:
- Utilizes nucleotide analog pulse labeling and DNA content-based cell sorting.
- Employs click chemistry for biotinylation followed by DNA fragmentation.
- Includes on-bead sequencing library generation for next-generation sequencing analysis.
Main Results:
- Successfully adapted Repli-seq using biotinylation for accurate RT measurement.
- Developed a comprehensive protocol compatible with various proliferating cell types.
- Demonstrated compatibility with high-throughput automation.
Conclusions:
- BioRepli-seq offers a robust and versatile approach for studying DNA replication timing.
- The protocol facilitates genome-wide RT analysis in diverse cellular contexts.
- This method advances the study of genome organization and replication dynamics.
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