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Related Concept Videos

The DNA Replication Fork01:02

The DNA Replication Fork

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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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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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Replication in Prokaryotes01:32

Replication in Prokaryotes

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DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
Many Proteins Work Together to Replicate the Chromosome
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The Replisome03:01

The Replisome

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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
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Replication in Eukaryotes01:29

Replication in Eukaryotes

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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.
Many Proteins Orchestrate Replication at the Origin
Eukaryotic replication follows many of the same...
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Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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Related Experiment Video

Updated: Jul 9, 2025

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

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Visualizing DNA replication by single-molecule analysis of replicated DNA.

Advaitha Madireddy1, Jeannine Gerhardt2

  • 1Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA; Department of Pediatrics Hematology/Oncology, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA.

STAR Protocols
|December 4, 2023
PubMed
Summary

This study details a protocol for Single-Molecule Analysis of Replicated DNA (SMARD), enabling visualization of DNA replication dynamics at specific genomic sites with high resolution.

Keywords:
Cell BiologyIn Situ HybridizationMicroscopyMolecular BiologySingle Cell

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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
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Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay
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Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization
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Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA replication is a fundamental biological process crucial for cell division and genetic stability.
  • Understanding DNA replication dynamics at specific genomic regions is essential for deciphering complex cellular mechanisms.

Purpose of the Study:

  • To present a detailed protocol for visualizing DNA replication using Single-Molecule Analysis of Replicated DNA (SMARD).
  • To enable high-resolution analysis of various stages of DNA replication, including initiation, progression, termination, and fork stalling.

Main Methods:

  • The protocol involves pulse labeling of DNA followed by isolation and stretching of genomic DNA.
  • Detection of replication is achieved through immunostaining and fluorescence in situ hybridization (FISH).
  • This method allows for single-molecule resolution visualization of DNA replication events.

Main Results:

  • SMARD provides a unique technique for visualizing DNA replication at specific genomic regions.
  • The protocol allows for the observation of replication initiation, progression, termination, and fork stalling events.
  • High-resolution imaging of DNA replication is achievable.

Conclusions:

  • The presented SMARD protocol offers a powerful tool for studying DNA replication mechanisms at the single-molecule level.
  • This technique facilitates detailed investigation into the dynamics of DNA replication forks and associated phenomena.
  • The protocol aids in understanding genomic stability and cellular responses to replication stress.