Assessing PCR Inhibition from Humic Substances

Abstract

Inhibition remains the greatest methodological challenge in molecular analysis of buried biological remains. Inhibitory compounds associated with soil environments comprise primarily of humic acids and fulvic acids, collectively referred to as humic substances. We examined the sensitivity of 13 DNA polymerases to both humic acids (11ng-110� g) and fulvic acids (9.4ng-94� g) and the concentration at which successful amplification can be achieved. This research identified that all 13 DNA polymerases tested exhibited inhibition with varying concentrations of humic acids and that 5 out of the 13 DNA polymerase tested exhibited inhibition with varying concentrations of fulvic acid. The most tolerant DNA polymerase to inhibition due to the presence of humic and fulvic acids is pfu DNA polymerase followed by KlenTaq ® LA DNA polymerase and RealTaq DNA polymerase that were both only inhibited by 11� g and 110� g of humic acids. In addition, we present the use of size exclusion chromatography to remove small molecular weight humic substance, dramatically increasing the success of molecular analysis on material associated with burial. This research has implications to the fields of environmental microbiology, soil science, forensic science and archaeological science.