What is The DNA fiber technique and how is it performed?

[Y Soun]

What is The DNA fiber technique and how is it performed?

[Fazil Hussein]

The DNA fiber technique, also known as DNA fiber analysis or DNA fiber spreading, is a powerful method used to study DNA replication and other DNA-related processes at the single-molecule level. This technique allows researchers to visualize and analyze the dynamics of DNA replication, including replication fork progression, origin firing, and fork stalling.

How It’s Performed:

1. Labeling DNA: Cells are first incubated with nucleotide analogs that can be incorporated into newly synthesized DNA. These analogs are often halogenated nucleotides, such as BrdU (bromodeoxyuridine) or IdU (iododeoxyuridine).

2. Lysing Cells: The cells are then lysed to release the DNA. This step is crucial to ensure that the DNA fibers are intact and can be spread out for analysis.

3. Spreading DNA: The DNA is spread onto a glass slide. This can be done using various techniques, such as the “DNA combing” method, where the DNA fibers are stretched and aligned on the slide.

4. Fixing and Staining: The DNA fibers are fixed onto the slide and stained with fluorescent antibodies that specifically bind to the incorporated nucleotide analogs. This allows the newly synthesized DNA to be visualized under a fluorescence microscope.

5. Imaging and Analysis: The stained DNA fibers are imaged using a fluorescence microscope. The images are then analyzed to measure various parameters of DNA replication, such as the length of replication tracks, the distance between replication origins, and the speed of replication fork progression.

This technique provides valuable insights into the mechanisms of DNA replication and the factors that influence it, such as DNA damage, replication stress, and the activity of replication proteins.

 

[A. Hammouda]

This is a useful open access article:

The DNA fibre technique – tracking helicases at work – ScienceDirect

 

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