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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
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In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
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OpenEMMU: A versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics.

Osvaldo Contreras1,2, Chris Thekkedam1, John Zaunders3

  • 1Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.

Iscience
|September 8, 2025
PubMed
Summary
This summary is machine-generated.

OpenEMMU is a new, affordable, open-source method for tracking DNA replication using 5-Ethynyl-2'-deoxyuridine (EdU) click chemistry. It improves staining efficiency and multiplexing for diverse cell types and biological specimens.

Keywords:
BiochemistryCell biologyComputational bioinformaticsDevelopmental biology

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Developmental Biology

Background:

  • 5-Ethynyl-2'-deoxyuridine (EdU) is crucial for DNA replication and cell cycle analysis via click chemistry.
  • Commercial EdU kits are expensive, proprietary, and have limited multiplexing and specimen compatibility.

Purpose of the Study:

  • To develop an optimized, affordable, and user-friendly EdU click chemistry platform.
  • To enhance EdU staining efficiency, brightness, and multiplexing capabilities.
  • To enable high-resolution imaging of DNA synthesis in various biological contexts.

Main Methods:

  • Developed OpenEMMU, an open-source platform using off-the-shelf reagents for EdU detection.
  • Utilized non-conjugated and conjugated antibodies for multiplexed staining.
  • Applied the method to diverse cell types, including T cells, embryonic tissues, organoids, and zebrafish larvae.

Main Results:

  • OpenEMMU demonstrated enhanced efficiency, brightness, and multiplexing for EdU staining.
  • Successfully imaged nascent DNA synthesis in developing embryos, organs (heart, forelimbs), and cardiac organoids.
  • Enabled deep-tissue 3D imaging of DNA synthesis in zebrafish larvae and under replication stress with high spatial resolution.

Conclusions:

  • OpenEMMU provides a flexible and precise platform for studying DNA replication dynamics.
  • This methodology advances research in organismal development, cell proliferation, and DNA synthesis.
  • Offers a cost-effective and versatile alternative to commercial EdU kits.