Ultrasensitive and Rapid Screening of Tumor-Associated Extrachromosomal Circular DNA by Selective Real-Time Rolling Circle Amplification

Analytical challenges in detecting sequencing-unknown extrachromosomal circular DNA (eccDNA)─a molecule with critical roles in tumorigenesis and therapeutic potential─primarily arise from interference by long linear genomic and mitochondrial DNA. Here, we for the first time introduce a selective real-time rolling circle amplification (sRT-RCA) method that overcomes this limitation by combining RecBCD and PacI dual-enzyme digestion to remove interfering nucleic acids, thus permitting direct Phi29 polymerase-mediated RT-RCA without the need for conventional physical purification step. This approach achieves unprecedented sensitivity (2 pg, ∼4 × 10⁻¹⁹ mol circular DNA molecules) within a 10⁴-fold higher background of genomic DNA. Furthermore, the established method, utilizing a simplified direct-lysis workflow, is capable of detecting eccDNA from as few as 125 cells. By this approach, we estimated that eccDNA averages 540 copies (8kb plasmid equivalents) in certain cancer cells (K562). Clinically, eccDNA was detected in 3/10 early stage and 6/10 advanced-stage patients. These findings indicate that eccDNA is widespread in cancer patients and demonstrate the potential utility of our method in clinical applications and translational research, thereby bridging a key technical gap and accelerating the translation of eccDNA-based diagnostics and personalized therapy.