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

Abstract

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 dodecyl sulfate [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 or phenol 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 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 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 gel electrophoresis, 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.

FIG. 1

Comparison of DNA fragment size distributions and relative DNA yields obtained with the nine DNA extraction procedures described in Table 2. Five-microliter portions from three replicate extractions were pooled, digested with RNase, and electrophoresed on a 0.8% (wt/vol) agarose gel. The results obtained for wetland sediment are shown. DNA extracts obtained from forest and agricultural soils produced similar results. Lane M contained 500 ng of λ-HindIII marker.

FIG. 2

Effects of Mini Bead Beater homogenization speed and duration on DNA yield and maximum DNA fragment size. (A) Influence of homogenization speed on DNA yield and fragment size when DNA extraction procedure 1 was used. (B and C) Influence of homogenization time on DNA yield (B) and maximum fragment size (C) at two homogenization speeds (3,300 and 3,800 rpm) when DNA extraction procedure 3 was used. The error bars indicate standard errors (n = 4). gdw, gram (dry weight).

FIG. 3

Effects of Mini Bead Beater-8 homogenization speed and duration and extraction protocol on DNA yield and maximum DNA fragment size. (A) Influence of homogenization speed on DNA yield and fragment size when DNA extraction procedure 1 was used. (B and C) Influence of homogenization time on DNA yield (B) and maximum fragment size (C) at two homogenization speeds (2,510 rpm [open symbols] and 2,670 rpm [solid symbols]) when DNA extraction procedure 1 (circles) and procedure 3 (triangles) were used. The error bars indicate standard errors (n = 4). gdw, gram (dry weight).