The comet assay: A contemporary approach for detecting genomic instability

The comet assay has evolved into a high-resolution, multifunctional technique for evaluating DNA damage, repair capacity, and epigenetic modifications. Over the past fifteen years, significant advancements-including the enzyme-modified comet assays (EMCA), Comet-FISH, and high-throughput platforms have substantially expanded its analytical capabilities. Specialized formats like the Flash comet assay and the BrdU comet assay further enhance the discrimination of DNA strand break types and replication-associated damage. Despite these innovations, issues related to standardization, reproducibility, and inter-laboratory consistency remain. Initiatives such as the Minimum Information for Reporting Comet Assay (MIRCA) guidelines, alongside the integration of automated imaging and machine learning, are being implemented to address these challenges. With its growing applications in environmental toxicology, biomonitoring, and clinical research, the comet assay is increasingly recognized as a key tool in precision toxicology and personalized medicine. This review highlights major technical developments and emerging applications of the comet assay over the past fifteen years, discusses sources of experimental variability and strategies for optimization, and provides an update on current laboratory protocols for assessing DNA damage induced by genotoxic agents.