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

Journal article, 2025

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 (SD3), which uses a robust method for finding DNA molecules and estimating the number of dots along each molecule. We have validated SD3 by comparing the outcome to manual analysis using DNA extracted from cells exposed to H2O2 as a model system. Our results show that SD3 achieves high accuracy and reduced analysis time relative to manual counting. SD3 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.