Accurate Detection and Quantification of Human Nuclear DNA

Accurate detection and quantification of human nuclear DNA (nDNA), named the human accelerated region 1 (HAR1) assay, was developed to simultaneously prove the human origin of forensic biological samples and optimize nDNA input for PCR in human DNA profiling. The HAR1 assay, a TaqMan probe-based real-time PCR assay targeting HAR1 in nDNA, was exclusively specific to human DNA samples with no cross-reactivity across 23 vertebrate species (mammals including non-human primates, birds, reptiles, amphibians, and fishes). This assay is capable of proving the human origin of the samples from as little as the DNA content of a single somatic cell. Moreover, accurate quantification of human nDNA was achieved down to the minimum input concentration (33 pg/μL) recommended by commercial DNA profiling kits. Furthermore, it was confirmed that the presence of non-human DNA mixed with human DNA did not interfere with the accuracy of human nDNA quantification. Therefore, the HAR1 assay represents a valuable and practical tool for improving both the accuracy and efficiency of human DNA profiling, particularly in forensic casework where sample quantity and quality may be limited and the presence of non-human DNA can complicate analysis.