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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1999, p. 4715–4724 0099-2240/99/$04.00 0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 65, No. 11

Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples
D. N. MILLER,* J. E. BRYANT,† E. L. MADSEN,
AND

W. C. GHIORSE

Section of Microbiology, Division ofBiological Sciences, Cornell University, Ithaca, New York 14853-8101
Received 1 July 1999/Accepted 6 August 1999

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecylsulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform orphenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion 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 physicallysis 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 homogenizers. 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 gelelectrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for 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. Our results indicate that for these types of samples, optimum DNA recovery requires brief,low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification. In the past decade, applications of molecular biological approaches have provided unique insights into the uncultured microbial communities of soils and waters because they avoid biases inherent in traditional culture-based microbiological methods (16). Thevalidity of using molecular techniques in environmental studies depends on obtaining representative extracts of nucleic acids from entire microbial communities. Nucleic acid extraction methods, however, suffer from compounded inefficiencies in the individual component steps, including incomplete cell lysis, DNA sorption to soil surfaces, coextraction of enzymatic inhibitors from soil, and loss,degradation, or damage of DNA. Thus, studies of DNA extraction techniques (14, 23, 24, 27) have indicated that these techniques can introduce biases of their own. In the initial efforts to extract DNA from sediments and soils workers used either cell extraction (recovery of cells from the soil matrix prior to cell lysis) or direct lysis within the soil matrix (13, 29, 35). Direct lysis techniques,however, have been used more because they yield more DNA and presumably a less biased sample of the microbial community diversity than cell extraction techniques yield (13, 21, 35). A major drawback of direct lysis methods is that more PCR-inhibitory substances are extracted along with the DNA (29, 36, 39). In addition, the number and diversity of the direct lysis DNA extraction protocols used for...