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Mechanistic insights into melanin-induced PCR inhibition and its NanoPCR-based mitigation
Kamayani Vajpayee1, Shriyansh Srivastava2, Shivkant Sharma3
1Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Central Campus, Navrangpura, Ahmedabad, Gujarat, 380009, India.
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.
Insights
Melanin inhibits PCR in forensic DNA typing by binding to Taq polymerase. Bovine serum albumin (BSA) and BSA-coated gold nanoparticles (AuNPs) effectively restore PCR, with BSA showing optimal results and BSA-coated AuNPs offering consistent facilitation.
Area of Science:
- Forensic Science
- Molecular Biology
- Biochemistry
Background:
- Melanin inhibits polymerase chain reaction (PCR), a critical technique in forensic DNA typing.
- This inhibition leads to allele dropout and reduced peak heights, compromising DNA analysis.
- Traditional methods for mitigating melanin inhibition are often unsuitable for low-template DNA samples.
Purpose of the Study:
- To elucidate the mechanism of melanin-induced PCR inhibition using computational methods.
- To evaluate the efficacy of different facilitators, including gold nanoparticles (AuNPs), BSA-coated AuNPs, and standard bovine serum albumin (BSA), in overcoming melanin inhibition.
- To identify optimal methods for enhancing PCR amplification in the presence of melanin for forensic applications.
Main Methods:
- Molecular docking and dynamics simulations to identify melanin's interaction with Taq polymerase.
- Short tandem repeat (STR) typing of melanin-inhibited samples treated with various facilitators.
- Comparative analysis of total peak height (TPH) and heterozygous peak height ratio (PHR) to assess restoration efficacy.
- Statistical analysis to determine the significance of enhancement provided by different facilitators.
Main Results:
- Melanin was found to interact with catalytic residues TYR671 and PHE667 of Taq polymerase, explaining its inhibitory effect.
- STR typing showed significant allele loss and reduced peak heights in melanin-treated samples.
- Standard BSA provided the highest PCR restoration (TPH = 786,122.7 RFU, mean PHR = 0.92), followed by BSA-coated AuNPs (TPH = 682,938.3 RFU, mean PHR = 0.90).
- BSA-coated AuNPs offered more consistent facilitation at lower concentrations compared to BSA.
Conclusions:
- Melanin inhibits PCR by directly interfering with Taq polymerase function.
- Both BSA and BSA-coated AuNPs are effective in restoring PCR amplification in the presence of melanin.
- BSA-coated AuNPs present a promising nanotechnology-based approach for consistent and reliable forensic DNA typing with melanin-inhibited samples.
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