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Abstract
Environmental DNA (eDNA) is a complex mixture of genomic DNA from many different organisms that can persist in soil from several months to years. In the soil environment, eDNA is subjected to a dynamic range of factors that determine its fate, including binding to soil minerals and humics, degradation by microbial DNases, and incorporation into the bacterial genome through transformation, conjugation, and transduction. The current dissertation aims to improve our understanding of the abiotic processes controlling the persistence and functionality of eDNA in the subsurface environment. While previous studies have typically relied on non-specific detection methods (fluorescent and UV-Vis detection) for evaluating gross DNA sorption to soil minerals, quantitative polymerase chain reaction (qPCR) was used to evaluate DNA partitioning based on the detection of a short, specific plasmid insert (116 base pairs). Subsequent genetic transformation associated with an antibiotic resistance gene (ARG) was then used to verify residual plasmid gene function. In a series of batch experiments using UV-Vis and qPCR detection methods, goethite sorbed more linear and plasmid eDNA than kaolinite or subsurface soil, suggesting that a small amount of goethite likely plays a disproportionate role in mineral sorption of DNA in soils. Observed shifts in the zero point of charge for the mineral sorbents in the presence of DNA indicated a specific adsorption mechanism with the sorbed species altering the surface properties of the sorbent. The higher DNA sorption capacity displayed by goethite when compared to kaolinite was attributed to goethite’s higher reactive site density and higher ZPC, which was more effective at sorbing anionic DNA under the test conditions. In terms of gene functionality, plasmid desorption was necessary for genetic transformation to occur, with transformation efficiency negatively correlated with the binding affinity of the minerals and soils. Moreover, the sorption of plasmids on goethite and kaolinite negatively affected the transformation ability of the ARG without altering the integrity of the smaller inserted gene used as a plasmid tracer for qPCR analysis. Taken together, DNA sorption and transformation by soil minerals was influenced by mineral type and qPCR was found to be an efficient tool for analyzing the persistence of specific genetic sequences, but did not verify subsequent gene function.