| Molecular biology techniques have been used in soil microbiology with the objective of enhancing the knowledge about the diversity, function, survival, and genetic transformations of introduced or indigenous microorganisms. These techniques require nucleic acids to be efficiently extracted and relatively intact (high MW) from microorganisms. Two main constraints occur with DNA extraction. One is the extract's contamination with humic substances that impair DNA quantification and biochemical manipulations. Purification procedures are time-consuming and significantly decrease DNA yields. The other concern is whether DNA is extracted effectively from the wide variety of soil organisms. A universal and efficient method for DNA extraction from soil microorganisms is not available, and information about recovery from the variety of microbes is lacking. The present studies examined approaches for humic substance elimination during extraction without strong solvents (phenol and/or chloroform). The modified method was tested with a variety of soils and compost and consistently gave high DNA yields with low humic substance contamination. Additional tests were done to evaluate DNA extraction from indigenous soil microorganisms by hybridization to phylogenetic probes targeting Bacteria, Archaea, and Eucarya domains as well for specific sub-groups as Gram-negatives (Proteobacteria) and Gram-positives (High G+C content). These analyses allowed detection of shifts in the composition of microbial soil population with regard to the Bacteria and Eucarya domain. Effects were also detected on specific Bacteria sub-groups. |
