Mechanistic Insights into Melanin-Induced PCR Inhibition and Its NanoPCR-Based Mitigation

Melanin is a potent inhibitor of PCR that inhibits forensic DNA typing by binding Taq polymerase and interfering with its function, resulting in allele dropout and decreased peak heights. Traditional mitigation measures, such as dilution or replacement of the polymerase, tend to result in loss of DNA and are not appropriate for low-template forensic samples. In this study, melanin-induced inhibition was explored by using molecular docking and dynamics simulation, which identified stable interaction with catalytic residues TYR671 and PHE667 (Kd = 31.76 ± 0.02 µM), interfering with polymerase function. Against this, gold nanoparticles (AuNPs) and BSA-coated AuNPs were employed as in situ facilitators. STR profiling established that BSA-coated AuNPs restored amplification efficiency significantly, with 2-fold improvement and recovery of heterozygous peaks. These results provide mechanistic insight into melanin inhibition and establish a nanotechnology-based approach to improve PCR results in recalcitrant forensic and clinical samples.