Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling

Area of Science:

• Molecular Biology
• Genotoxicology
• Biochemistry

Background:

• The comet assay detects DNA strand breaks after genotoxic agent exposure.
• Conventional comet assays with DNA stains cannot differentiate between DNA single-strand breaks (SSBs) and double-strand breaks (DSBs).
• Distinguishing between SSBs and DSBs is crucial as DSBs pose a greater cellular risk.

Purpose of the Study:

• To develop a method for differentially identifying SSBs and DSBs within the same comet.
• To introduce the polymerase-assisted DNA damage analysis (PADDA) protocol for enhanced DNA damage detection.

Main Methods:

• Developed the polymerase-assisted DNA damage analysis (PADDA) protocol.
• Combined PADDA with the Flash comet protocol or applied it to fixed cells.
• Utilized DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides.
• Tested the protocol on TK6 or HaCat cells exposed to hydrogen peroxide, X-rays, or DNA cutting enzymes.

Main Results:

• PADDA successfully differentiated between SSBs and DSBs in treated cells.
• The protocol demonstrated a wider detection range compared to conventional methods.
• PADDA unveiled previously undetected DNA strand breaks.

Conclusions:

• PADDA is an effective method for distinguishing and quantifying SSBs and DSBs in the comet assay.
• This technique provides a more comprehensive analysis of DNA damage.
• PADDA enhances the sensitivity of DNA damage detection in genotoxicity studies.