Work

Stained DNA Dot Detection (SD3): An automated tool for quantifying fluorescent features along single stretched DNA molecules

Year: 2025

Type: article

Abstract: The main information in DNA is its four-letter sequence that builds up the genetic information and that is traditionally read using sequencing methodologies. DNA can, however, also carry other important information, such as epigenetic marks and DNA damage. This information has recently been visualized along single DNA molecules using fluorescent labels. Quantifying fluorescent labels along DNA is done by counting the number of “dots” per length of each DNA molecule on DNA stretched on a glass surface. So far, a major challenge has been the lack of standardized data analysis tools. Focusing on DNA damage, we here present a Matlab-based automated software, Stained DNA Dot Detection (SD 3 ), which uses a robust method for finding DNA molecules and estimating the number of dots along each molecule. We have validated SD 3 by comparing the outcome to manual analysis using DNA extracted from cells exposed to H 2 O 2 as a model system. Our results show that SD 3 achieves high accuracy and reduced analysis time relative to manual counting. SD 3 allows the user to define specific parameters regarding the DNA molecule and the location of dots to include during analysis via a user-friendly interface. We foresee that our open-source software can have broad use in the analysis of single DNA molecules and their modifications in research and in diagnostics. • We developed Stained DNA Dot Detection (SD 3 ), an open source automated tool for analysing fluorescent labels on stretched DNA. • SD 3 can be used to investigate damage on stretched individual molecules. • SD 3 has a user-friendly interface for easy definition of molecule and dot parameters. • SD 3 performance correlates strongly with manual measurement and allows large sets of images to be analyzed in minutes. • Manual evaluation of DNA length and fluorescent labels on stretched DNA is subjective.

Source: DNA repair

DOI: 10.1016/j.dnarep.2025.103836