Extraction and purification of microbial DNA from soil and sediment samples

Abstract

Knowledge of the microbial diversity in natural ecosystems has long been limited because only a minority of naturally occurring microbes can be cultured using standard techniques. Several protocols for the extraction of nucleic acids directly from the environmental matrix have been recently developed to circumvent this problem and this review covers the major extraction procedures currently used to obtain microbial DNA from environmental samples. DNA extraction procedures can involve cell extraction or direct lysis, depending on whether or not the microbial cells are isolated from their matrix. An extraction protocol generally comprises three steps: cell lysis that can be chemical, mechanical and enzymatic, removal of cell fragments and nucleic acid precipitation and purification. Direct lysis methods are more often used than cell extraction ones because they are less time consuming and give a better recovery, resulting in an extracted DNA more representative of the whole microbial community present in the sample. However, with direct lysis, contaminants are also extracted which interfere with the DNA extract. As a consequence, a more extensive purification step is required. At least four types of purification are commonly used: cesium chloride density gradient ultracentrifugation, chromatography, electrophoresis and dialysis and filtration. To remove all contaminants, it could be recommended that several purification procedures be combined, depending on the environmental matrix. The efficiency of extraction/purification depends on the properties of the environmental sample, and each step of the extraction procedure must be adjusted for each sample. Moreover, each step of the procedure suffers from shortcomings, and each additional step inevitably induces a DNA loss. Thus, the choice of a protocol must be a compromise between the recovery of DNA that will be the most representative of the microbial community and the quality of the DNA obtained that is imposed by the objectives of the work, such as detection of specific organisms or assessment of the total microbial community structure. Nevertheless, molecular techniques, that could be used in combination with cultivation techniques, are powerful methods for surveying the microbial diversity in environmental samples, although investigators must be aware that such techniques are not exempt of methodological biases.