STR-Uggles: Overcoming Humic Acid Inhibition Using Combined STR & qPCR Kit Chemistries

DNA profiling can fail, or produce poor results, when naturally occurring materials are present during the amplification step. This study demonstrates that simple modifications to the reaction setup can overcome this obstacle. PCR inhibition is caused by a range of compounds including haem, humic acid and dyes. Various strategies to overcome this inhibitory effect have been explored, such as improving extraction methods to remove these compounds, diluting samples to reduce inhibitor concentration, or using inhibitor-tolerant DNA polymerases. In this study, we evaluate whether modified setups can help mitigate the effects of humic acid, a common inhibitor that induces various inhibition mechanisms.

We combined the GlobalFiler STR kit with Investigator Quantiplex Pro into a single reaction. Supplementing the amplification GlobalFiler with additional reagents creates altered amplification environments that use additional DNA polymerase and reaction buffer.

The modified setups outperformed the standard GlobalFiler protocol, even at the highest concentration of humic acid tested. The STR reactions supplemented with qPCR reagents produced higher-quality profiles with improved allele amplification and an even peak balance, indicating that a dual-DNA polymerase system offers a more robust and inhibitor-tolerant environment for STR amplification. In addition to demonstrating the value of this combined approach, these data provide a comprehensive dataset characterising the impact of increasing humic acid concentrations on profile quality from an ideal DNA input.

For PCR inhibitors with similar mechanisms this approach offers broader applicability in forensic casework and a promising step toward more reliable and robust profiling of inhibited samples.