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Related Concept Videos

Lagging Strand Synthesis01:59

Lagging Strand Synthesis

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During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
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Chromosome Replication02:31

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Before a cell can divide, it must accurately replicate all of its chromosomes, including the DNA and its associated histone and non-histone proteins.  This process begins at numerous origins of replication during the S phase of the cell cycle in each of a cell’s chromosomes simultaneously. Certain nucleotides can act as origins of replication, but these sequences are not well defined - especially in complex, multi-cellular, eukaryotic species. The length of DNA that spans an origin...
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Related Experiment Video

Updated: Sep 8, 2025

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization
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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.

STAR Protocols
|September 5, 2025
PubMed
Summary

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.

Keywords:
Cell BiologyGeneticsGenomicsMolecular Biology

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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.