Soil Chemical Factors

Among the key chemical factors that may interfere with molecular analysis of soil communities are soil constituents with cation and anion exchange capacity, such as humic acids, clays, and soil organic matter (SOM). Clay minerals and SOM possess a net negative charge. Individual, negatively charged clay particles in a moist soil will be surrounded by hydrated cations, which create a localized zone of positive charge. This will attract microorganisms, which possess a net negative charge at the pH of most soil habitats. Binding of bacteria to solid surfaces through such ionic interactions makes it difficult to separate both cells and DNA/RNA released from cells from the soil matrix. Soil type has a significant influence on

DNA/RNA extraction efficiency. Nucleic acid extraction may be particularly problematic from soils with high clay, organic matter, and/or humic contents. Effective removal of humic acids is often required prior to quantifying or amplifying DNA.

Humic substances inhibit Taq DNA polymerase in the PCR, interfere with restriction enzyme digestion, and reduce transformation efficiency during cloning and DNA hybridization specificity. Humic substances are difficult to remove as they remain soluble under conditions similar to those of DNA; hence, direct extraction of DNA may require an additional purification step to obtain DNA of sufficient purity for downstream assays. Use of polyvinylpyrrolidone may help to remove SOM from the cell preparations. Subsequent cesium chloride density gradient cen-trifugation yields DNA of high quality. Despite their effectiveness, these procedures are too labor intensive for use in large experiments. DNA extraction kits are available that include improved DNA clean-up steps and yield higher quality DNA extracts with fewer impurities that affect downstream DNA analyses.

The presence of humic acids and SOM also interferes with fluorescence microscopy. Nonspecific background fluorescence caused by binding of dyes to charged particles makes it difficult to resolve and quantify soil microorganisms. If microscopic images will be subject to image analysis, it also becomes difficult to "train" the software to separate cells from inorganic particles and produce an accurate count.

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