Mechanistic insights into melanin-induced PCR inhibition and its NanoPCR-based mitigation.

Melanin is a potent inhibitor of PCR, which interferes with forensic DNA typing by binding to Taq polymerase and disrupting its function, leading to allele dropout and decreased peak heights. Traditional mitigation measures, such as dilution and replacement of the polymerase, often result in the loss of DNA and are not suitable for low-template forensic samples. In this study, melanin-induced inhibition was explored using molecular docking and dynamics simulation, which identified stable interaction with catalytic residues TYR671 and PHE667 (Kd = 31.76 ± 0.02 µM), thereby interfering with polymerase function. STR typing of the inhibitor-treated sample showed a total allelic loss of SE33 and Penta E, dropout at D12S391, and substantially reduced peak heights. The overall peak height was 443,409.3 RFU. Facilitating methods using AuNPs, BSA-coated AuNPs, and standard BSA were further compared. AuNPs provided partial restoration (TPH = 545,605.7 RFU), whereas BSA-coated AuNPs provided improved restoration (TPH = 682,938.3 RFU) with harmonious heterozygous peaks (mean PHR = 0.90). Standard BSA had the highest restoration (TPH = 786,122.7 RFU; mean PHR = 0.92), restored alleles between dye channels. Statistical analysis revealed significant enhancement by BSA (p < 0.0001) and moderate enhancement by BSA-coated AuNPs (p = 0.0186). The BSA yielded optimal results, but it also exhibited larger sample differences. On the other hand, BSA-coated AuNPs offered more consistent facilitation at a very low concentration compared to BSA. This study thus explains the mode of PCR inhibition by melanin. It further demonstrates that BSA remains the most effective facilitator, but it also has its own limitations. In contrast, BSA-coated AuNPs offer a reliable nanotechnology-based method to overcome this limitation in forensic PCR applications.