A novel droplet digital PCR method for assessing the quantity and quality of degraded samples

Accurate evaluation of degraded crime scene samples is critical for forensic DNA analysis, as the concentration and integrity of DNA in samples are directly related to the success rate of DNA typing. However, current degradation assessment methods commonly used in forensic laboratories have certain limitations, especially when applied to highly degraded samples. Droplet digital PCR (ddPCR) achieves absolute DNA quantification through physical partitioning, offering exceptional sensitivity, reproducibility, and stability, and provides distinct advantages for degraded DNA analysis. We developed a triplex ddPCR system capable of simultaneously detecting three DNA fragments of different lengths (75 bp, 145 bp, and 235 bp), and proposed calculating degradation ratios (DRs) to precisely quantify DNA degradation levels. Results demonstrate high sensitivity, reliably detecting DNA degradation in samples with as few as two copies. Based on DR values, we suggest classifying degradation as mildly to moderately degraded, highly degraded, and extremely degraded, establishing a tiered assessment framework. This system enables forensic laboratories to rapidly evaluate DNA degradation severity, guide subsequent analytical workflows, inform optimal processing strategies, and support both evidence interpretation and the development of new techniques for evaluating degraded DNA.