Palao Cécile, Kovacs Adèle, Teixeira Maria Teresa, Richard Guy-Franck
Département Génomes & Génétique, Institut Pasteur, Université de Paris, CNRS UMR 3525, 25 rue du Dr Roux, 75015, Paris, France.
Sorbonne Université, Collège Doctoral, 75005, Paris, France.
Biol Methods Protoc. 2025 Aug 9;10(1):bpaf059. doi: 10.1093/biomethods/bpaf059. eCollection 2025.
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.
DNA双链断裂（DSBs）是基因组完整性中的关键事件，由内源性细胞过程和外源性因素引起。这些断裂与各种基因组畸变和染色体重排有关，导致癌症和遗传疾病。常见和罕见的脆性位点包含重复元件和非B型DNA结构，在复制应激下特别容易断裂，这在癌症发展和遗传疾病中起关键作用。在微卫星或非B型DNA结构等重复序列背景下准确量化DNA断裂在技术上具有挑战性。我们一直在比较四种不同的方法来可靠地量化重复DNA中的DSBs，即Southern印迹法、DSB-PCR、实时DSB-qPCR和数字PCR（dPCR）。我们在此表明，与其他方法相比，dPCR具有更高的灵敏度和特异性。这为未来的疾病诊断、理解产生染色体断裂的分子机制以及开发针对微卫星扩增疾病的基因疗法提供了重要应用。
