Fast and accurate quantification of double-strand breaks in microsatellites by digital PCR.

DNA double-strand breaks (DSBs) represent critical events in genome integrity, arising from both endogenous cellular processes and exogenous factors. These breaks are implicated in various genomic aberrations and chromosomal rearrangements, leading to cancers and genetic disorders. Common and rare fragile sites, containing repetitive elements and non-B DNA structures, are particularly prone to breakage under replication stress, which play a pivotal role in cancer development and genetic diseases. Accurate quantification of DNA breaks in the context of repetitive sequences such as microsatellites or non-B DNA structures is technically challenging. We have been comparing four different methods to reliably quantify DSBs in repetitive DNA, namely Southern blot, DSB-PCR, real-time DSB-qPCR, and digital PCR (dPCR). We show here that dPCR offers enhanced sensitivity and specificity compared to other methods. This provides significant applications for future disease diagnosis, understanding molecular mechanisms generating chromosomal breakage and for the development of gene therapies for microsatellite expansion disorders.