Extraction and purification of microbial DNA from soil and sediment samples

Knowledge of the microbial diversity in natural ecosystems has long been li mited because only a minority of naturally occurring microbes can be cultur ed using standard techniques. Several protocols for the extraction of nucle ic acids directly from the environmental matrix have been recently develope d to circumvent this problem and this review covers the major extraction pr ocedures currently used to obtain microbial DNA from environmental samples. DNA extraction procedures can involve cell extraction or direct lysis, depe nding on whether or not the microbial cells are isolated from their matrix. An extraction protocol generally comprises three steps: cell lysis that ca n be chemical, mechanical and enzymatic, removal of cell fragments and nucl eic 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 ex tracted DNA more representative of the whole microbial community present in the sample. However, with direct lysis, contaminants are also extracted wh ich interfere with the DNA extract. As a consequence, a more extensive puri fication 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, d epending 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 adj usted for each sample. Moreover, each step of the procedure suffers from sh ortcomings, and each additional step inevitably induces a DNA loss. Thus, t he choice of a protocol must be a compromise between the recovery of DNA th at will be the most representative of the microbial community and the quali ty 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 com munity structure. Nevertheless, molecular techniques, that could be used in combination with cultivation techniques, are powerful methods for surveyin g the microbial diversity in environmental samples, although investigators must be aware that such techniques are not exempt of methodological biases. (C) 2001 Elsevier Science B.V. All rights reserved.