Related Experiment Video
Updated: Sep 26, 2025
07:18
Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique
Published on: October 27, 2011
40.1K
DNA replication profiling by molecular combing on single DNA fibers.
1Developmental Therapeutics Branch, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA.
STAR Protocols
|April 25, 2022
Summary
This protocol tracks DNA synthesis at the single-molecule level, visualizing replication fork progression and cellular responses to replication stress for detailed DNA replication dynamics studies.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA replication is a fundamental biological process essential for cell division and genetic stability.
- Understanding DNA replication dynamics is crucial for comprehending various cellular processes and disease mechanisms.
Purpose of the Study:
- To present a protocol for profiling DNA synthesis at single-molecule resolution.
- To enable the study of DNA replication dynamics, cellular responses to replication stress, and replication fork progression.
Main Methods:
- Stretching DNA fibers uniformly on silanized coverslips.
- Tracing replicating DNA using thymidine analogs and specific antibodies.
- Visualizing single DNA fibers with an anti-single stranded DNA antibody.
Main Results:
- Enables single-molecule resolution profiling of DNA synthesis progression.
- Allows for the detailed study of DNA replication dynamics.
- Facilitates investigation of cellular responses to replication stress.
Conclusions:
- This protocol provides a powerful tool for dissecting DNA replication mechanisms at high resolution.
- When combined with fluorescent in situ hybridization, it allows for locus-specific analysis of replication fork progression.
- The method is valuable for studying DNA replication dynamics and stress responses in various biological contexts.
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