The Effects of Known PCR Inhibitors on Droplet Digital PCR Performance

This study is an analysis of how forensic samples may be affected by compounds that can inhibit the polymerase chain reaction (PCR). Inhibitors associated with forensic samples include substances that are endogenous with the sample (calcium, melanin), absorbed from the environment (humic acid, indigo dye)1, or introduced during laboratory processing (phenol1 , glove powder2). Human DNA quantification kits using quantitative real-time PCR (qPCR) instrumentation, the current standard for the quantification of DNA, are used to detect inhibitors in samples. Droplet digital PCR (ddPCR) relies on absolute quantification of positive and negative droplets and therefore may be less susceptible to inhibition than traditional qPCR techniques. Samples were prepared with a range of known inhibitor concentrations including aluminum (Al), copper (Cu), iron (Fe), and humic acid (HA). Metal ion concentrations ranged from 7 mM to 43 mM and Humic acid concentrations ranged from 166.67 ng/μL to 1000 ng/μL. Duplicate samples with 5 ng/μL of control DNA were used to determine the robustness of ddPCR in the presence of with inhibitors using the QX100 ddPCR system (BIO-RAD, Hercules, CA). Results indicate that iron and humic acid both show little effect on ddPCR: iron shows inhibition only with extreme inhibitor concentration. By contrast, copper slowly drops the amplitude of the positive droplets to the baseline with increasing inhibition. Aluminum drastically impacts ddPCR performance, resulting in positive droplets up to six times the amplitude of normal droplets in a scattered pattern, making the results unusable. These findings suggest that ddPCR may prove to be a more robust technique for inhibited forensic samples in some but not all settings.