Digital PCR in forensic genetics: principles, applications, and challenges

Digital PCR (dPCR) has emerged as a transformative technology in forensic genetics, offering absolute quantification of nucleic acids without the need for standard curves. This review comprehensively summarizes the principles, analytical advantages, and diverse applications of dPCR in forensic medicine, while also addressing current challenges and future perspectives. By partitioning samples into thousands to millions of individual reactions, dPCR enables high-precision quantification, exceptional sensitivity for rare variants, and robust tolerance to PCR inhibitors and degraded DNA-characteristics particularly advantageous for analyzing challenging forensic specimens such as trace, degraded, and inhibitor-laden samples. Key applications discussed include DNA mixture deconvolution, differentiation of monozygotic twins through low-frequency variant detection, forensic age estimation based on DNA methylation and other molecular markers, body fluid and tissue identification using RNA and DNA methylation markers, and species identification of non-human biological samples. Despite its significant potential, the widespread implementation of dPCR in routine forensic practice is constrained by limited multiplexing capacity, higher per-sample costs, and the absence of standardized forensic guidelines. Future advancements in microfluidic integration, multiplexing technologies, and portable platform development are expected to further expand the utility of dPCR, positioning it as a powerful complementary tool in forensic casework, particularly for complex and compromised samples.