Abstract
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.
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I declare that the authors have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper. The molecular dynamics simulation movies used in this manuscript have been uploaded to the public data repository Zenodo to ensure transparency and accessibility, as per FAIR data principles. The Zenodo DOI is: https://doi.org/10.5281/zenodo.13933345. These movies are not part of any previously published or pending manuscripts. They serve as supporting data for the molecular interaction insights discussed in this submission and are being referenced here for the first time in a peer-reviewed publication. The data sharing on Zenodo is intended solely to comply with data availability standards and does not constitute prior or dual publication.
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Acknowledgements
The authors gratefully acknowledge the Council of Scientific and Industrial Research (CSIR), Government of India, for awarding the Senior Research Fellowship (SRF) to Kamayani Vajpayee and Shriyansh Srivastava. The authors also thank Ahmedabad University, Indian Institute of Technology Gandhinagar, and the Central University of Gujarat, Gandhinagar, for providing laboratory space, infrastructure, and research resources essential for the successful completion of this study. We also thank Professor Souvik Sengupta, Associate Professor, School of Arts and Sciences, Ahmedabad University for his critics and suggestions that has improved the work presented.
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K.V. conceptualized the study, curated data, performed formal analysis, conducted investigations, developed methodology, contributed to validation and visualisation, and prepared figure 1, 3, and 4, wrote the original draft. S.S. (Shriyansh Srivastava) contributed to data curation, formal analysis, investigation, methodology, and writing of the original draft. S.S. (Shivkant Sharma) was involved in data curation. S.G. contributed to data curation, formal analysis, prepared figure 2, methodology, and writing of the original draft. A.S. participated in conceptualisation, data curation, formal analysis, investigation, methodology, validation, visualisation, and original draft writing. V.P. contributed to data curation. H.R.D. contributed to methodology, validation, and writing—review and editing. A.P. contributed to formal analysis, validation, and writing—review and editing. R.K.S. was involved in conceptualisation, data curation, formal analysis, investigation, methodology, validation, visualisation, and writing—review and editing. All authors reviewed the manuscript.
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Vajpayee, K., Srivastava, S., Sharma, S. et al. Mechanistic insights into melanin-induced PCR inhibition and its NanoPCR-based mitigation. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35010-w
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DOI: https://doi.org/10.1038/s41598-026-35010-w
