Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples

We compared and statistically evaluated the effectiveness of nine DNA extra ction procedures by using frozen and dried samples of two silt loam soils a nd a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], c hloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different p hysical disruption methods (bead mill homogenization and freeze-thaw lysis) , and lysozyme digestion were evaluated based on the yield and molecular si ze of the recovered DNA. Pairwise comparisons of the nine extraction proced ures revealed that bead mill homogenization with SDS combined with either c hloroform or phenol optimized both the amount of DNA extracted and the mole cular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digesti on before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different ho mogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophor esis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) fo r DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. O ur results indicate that for these types of samples, optimum DNA recovery r equires brief, low-speed bead mill homogenization in the presence of a phos phate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column pu rification.