Inhibition of DNA Polymerases Used in Q-PCR by Structurally Different Soil-Derived Humic Substances

Real-time PCR for the quantitative assessment of microbial genes in DNA extracted from environmental samples is increasingly being used in microbial ecology studies. A significant problem with the quantitative aspect of the method is the possible inhibition of the PCR process by humic substances co-extracted with the DNA. A comparison of the inhibition exerted by five structurally different humic substances on six commercially available DNA polymerases revealed large differences in the resistance of the polymerases to inhibition. Depending on the DNA polymerases (or their formulation) the addition of Bovine Serum Albumin (BSA) to the mastermix reaction generally increased the resistance to the different humic substances and decreased differences between the polymerases. One of the tested polymerases was clearly hampered by the addition of BSA, indicating that BSA cannot be added to just any mastermix to improve enzyme performance. The structural differences in the tested humic acids suggest that the mechanism of the inhibition is not a feature of all organic structures, but mainly related to the presence of certain phenolic or quinonoid structures.