INTRODUCTION

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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). The validity 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 soils and sediments are daunting (11-13, 20, 27, 29, 30, 44), but each protocol usually includes from one to all three of the following basic elements: physical disruption, chemical lysis, and enzymatic lysis.

Four different physical disruption techniques, freeze-thawing (8, 16, 20, 31, 38), bead mill homogenization (4, 20, 22, 27, 35), ultrasonication (30), and grinding under liquid nitrogen (41, 44), have been described, and freeze-thaw disruption and bead mill homogenization are the most common. It is well established that bead mill homogenization yields more DNA than freeze-thaw disruption yields (20, 21, 27, 33). The drawbacks to bead mill homogenization include the fact that larger amounts of contaminating humic acids are recovered (21, 29, 33) and the fact that, in some instances, the DNA is sheared (21). The chemical lysis procedures used in the methods that have been described also vary, but they lysis mixtures can be categorized into mixtures that contain detergent (either sodium dodecyl sulfate [SDS] [4, 8, 12, 14, 15, 20, 25, 28, 36-38, 44] or Sarkosyl [14, 34, 37]), mixtures that contain NaCl, and mixtures that contain various buffers (usually Tris or phosphate, pH 7 to 8). The modifications of the basic chemical lysis techniques include high-temperature (60°C to boiling) incubation (4, 20, 34, 35), a phenol (8, 33, 38) or chloroform (12) extraction step, and incorporation of chelating agents (EDTA and Chelex 100) to inhibit nucleases and disperse soil particles (6, 18). The efficacy of diverse chemical lysis components remains largely unknown since only overall DNA recovery after cell lysis and subsequent purification is reported. Furthermore, the cellular lysis efficiency at each step of a protocol is reported only rarely (27). A final component of many DNA extraction techniques is enzymatic lysis. Lysozyme (4, 8, 11, 14, 31, 36, 38), proteinase K (25, 36, 44), achromopeptidase (7), and pronase E (14) have all been employed to promote cell lysis, and lysozyme digestion is the most widely used procedure. Because of a lack of comparative studies, it is unclear what effect the addition of an enzymatic lysis step has on DNA yield.

An added layer of complexity when the DNA yields obtained with different protocols are compared is the DNA purification method employed. For most soils that contain high concentrations of humic acids, agarose gel electrophoresis (16, 22, 25, 27, 28, 43, 44), Sephadex G-200 column chromatography (7, 8, 12, 20, 31, 39), and silica-based DNA binding (5, 27, 44) have been used individually or in combination to separate humic acids and other enzyme inhibitors from DNA. Purification efficiency is usually judged by the amount of DNA recovered and the success of the methods used to remove contaminants that inhibit PCR enzymes and other enzymes required for molecular analyses.

While the strengths and weaknesses of the various complete extraction and purification methods have been discussed previously (17, 20, 21, 27, 35, 39, 44), in general detailed comparisons of individual elements of the methods (e.g., the effectiveness of extraction with and without a chelating agent) have not been performed. Meaningful comparisons are also confounded by the variety of sample types examined and by the variable analytical procedures used to quantify DNA extraction efficiency and purity. Thus, selection of an appropriate DNA extraction and purification procedure from among the procedures that have been described to date remains a major problem in the application of molecular techniques to studies of soil and sediment microbial communities.

The objectives of this study were to compare the most common elements of DNA extraction and purification protocols and to use the information obtained to develop a comprehensive method for obtaining whole-community DNA from soil and sediment samples containing different quantities of organic matter. Nine different extraction procedures with contrasting physical, chemical, and enzymatic elements were evaluated on the basis of the following criteria: (i) efficiency of cell lysis, (ii) total yield of DNA, and (iii) molecular sizes of the DNA fragments. Four purification procedures were also evaluated by testing for the presence of PCR-inhibitory substances and the loss of DNA during the procedures. Bead mill homogenization conditions were then optimized with two different homogenization instruments in order to obtain the largest amount of DNA with the smallest amount of DNA shearing. The result was an optimized direct lysis DNA extraction and purification protocol for soils and sediments having diverse organic matter contents.

	MATERIALS AND METHODS

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Soil and sediment sampling and preservation. Samples of a single type of soil (Collamer silt loam) from both an agricultural field and an adjacent forest and a wetland sediment that had the same silt loam texture but contained a different amount of organic matter (Table 1) were used in this study. The forest and agricultural soils were collected from McGowan Woods and an adjacent agricultural field (Cornell University, Ithaca, N.Y.), respectively. An ethanol-flamed spatula was used to collect approximately 400-g samples from the upper 2 to 5 cm of soil at each site. The soil samples were immediately transported to the laboratory in sterile, 200-ml Nalgene bottles. Approximately 400 ml of wetland sediment containing organic matter from decaying vegetation was collected at Sapsucker Woods (Ithaca, N.Y.) from the sediment-water interface by immersing and filling a previously sterilized 500-ml Nalgene bottle. The sample was immediately placed on ice and returned to the laboratory. Subsamples (approximately 100 g of soil or 200 ml of sediment) were set aside for use in chemical and particle size analyses. All of the samples used for DNA extraction were frozen at 20°C on the day of collection, lyophilized (Labconco Lyph-lock 4.5 lyophilizer), ground to a fine powder with a mortar and pestle, and stored at 20°C. All of the surfaces that contacted the soil were sterile or had previously been rinsed with a 10% (vol/vol) Clorox solution and then with sterile deionized water to remove any contaminating DNA.

Soil and sediment analysis. The texture, organic carbon content, and pH of the soils and sediment were determined by standard methods at the Cornell Soil Analysis Facility in the Department of Soil, Crop, and Atmospheric Sciences (Table 1). The total bacterial cell count for each sample was estimated by using an acridine orange epifluorescence direct counting method (2, 10) and a Zeiss standard epifluorescence microscope equipped with a Zeiss 09 filter combination.

Experimental design for DNA extraction procedures. A total of nine different procedures were evaluated by using each of the three sample types (Table 2). These procedures were designed to assess the effects of various combinations of SDS, organic solvents, chelation, enzymatic pretreatment with lysozyme, and bead mill homogenization or freeze-thaw lysis on DNA extraction and yield. Triplicate 50-mg samples of the wetland sediment and quadruplicate 100-mg samples of the agricultural and forest soils were added to 2-ml screw-cap plastic vials (Laboratory Products Sales, Rochester, N.Y.) containing 2 g of sterile, 0.1-mm-diameter zirconium-silica beads (BioSpec Products, Bartlesville, Okla.).

Enzymatic, chemical, and physical lysis treatments. The enzymatic pretreatments, chemical treatments, and physical lysis conditions used for the nine DNA extraction procedures are shown in Table 2. Because the ratio of liquid volume to sediment volume influences lysis efficiency, the extract liquid volume used for all extractions was 900 µl. Bead mill homogenization was used in procedures 1 through 4, 8, and 9 after the chemical treatments were performed (Table 2). The tubes were shaken for two 5-min periods at 1,600 rpm in a Mini Bead Beater (BioSpec Products). Three freeze-thaw cycles were used in procedures 5 through 7. Each cycle consisted of immersion for 2 min in liquid nitrogen, followed by immersion for 5 min in a 65°C water bath.

Recovery and purification of DNA. DNA released during each extraction procedure was recovered by the method of Herrick et al. (12). Briefly, beads and soil or sediment were separated from suspensions by centrifugation (10 s, 10,000 × g) with a microcentrifuge (model 235C; Fisher Scientific), and each liquid extract was transferred to a sterile 2-ml Eppendorf tube. The liquid remaining in the interstices of the bead bed was then collected by piercing the bottom of the tube with a hot 27-gauge needle and placing the pierced tube into a 15-ml, chloroform-safe, screw-cap polypropylene tube (Corning) containing a 1.5-ml Eppendorf tube with its top removed. The nested tubes were then centrifuged for 15 min (1,400 × g) with a tabletop centrifuge (model TJ6; Beckman). During centrifugation, liquid trapped in the bead bed drained into the lower 1.5-ml tube. This liquid was then pooled with the previously collected liquid from the sample.

Estimating cellular lysis in extracted sediment samples. The effects of the DNA extraction procedures on cellular lysis were estimated only with the wetland sediments, as follows. Triplicate 50-mg samples were subjected to each extraction procedure (Table 2) and the subsequent DNA recovery procedure as described above. Three formalin-preserved, freeze-dried samples were also processed from the DNA recovery step through the DAPI (4',6-diamidino-2-phenylindole) staining step and served as "no-DNA-extraction" controls that accounted for cell loss due to sample manipulation. The sediment and beads remaining after the crude DNA extract was removed were transferred to a 10-ml screw-cap tube containing 4 ml of phosphate-buffered saline (145 mM NaCl, 500 mM NaH₂PO₄; pH 7.4) and 50 µl of a 37% (vol/vol) formalin solution. The tube was shaken for 15 min at 200 rpm on a rotary shaker to disperse the sediment particles and bacteria. The tube was then vortexed for 15 s, and the beads were allowed to settle for 10 s. One milliliter of the supernatant was centrifuged for 10 min at 10,000 × g to pellet the bacteria, and the pellet was then resuspended in 1.0 ml of phosphate-buffered saline by vortexing. The wash step was necessary to remove any remaining phenol or chloroform from the sample, which would have preferentially absorbed the fluorescent dyes. A 5-µl drop of the resuspended sample was mixed with a 5-µl drop of a DAPI solution (0.05 mg of DAPI/ml of deionized water) on an ethanol-washed slide and covered with a 22-mm² coverslip. The total number of bacteria per milliliter of original suspension was determined by counting the number DAPI-stained blue fluorescent cells per microscopic field by using a Zeiss Standard 18 epifluorescence microscope equipped with a 100× Neofluar lens and an appropriate filter combination for DAPI. An area-to-volume calculation and appropriate dilution factors were used to convert the counting results to cells per milliliter and then to cells per gram (dry weight), as described previously (2). The efficiency of cell lysis for an extracted sample (expressed as a percentage) was calculated based on the difference between the bacterial cell count of the extracted preparation and the bacterial cell count of the no-DNA-extraction control.

Evaluation of DNA purification procedures. DNA extraction procedure 4 (SDS-Chelex 100 treatment with bead mill homogenization) was found to produce the most PCR-inhibitory extracts of all of the DNA extraction procedures tested with the PCR inhibition assay (see below). Therefore, DNA extracted by this procedure was used to test DNA purification procedures, as follows. A 500-mg forest soil sample, a 500-mg agricultural soil sample, and a 200-mg sediment sample were extracted as described above. The final volumes of the crude extracts were adjusted to 1.5 ml with sterile, distilled water. The RNA in each crude extract was digested by adding 150 µl of an RNase solution (10 mg of RNase per ml, 100 mM Tris HCl, 10 mM EDTA [pH 8]) and incubating the preparation for 1 h at 37°C. Subsamples (25 to 100 µl) of the RNase-digested soil and sediment extracts were purified in quadruplicate by using the four methods (methods i to iv) described below.

(i) Method i. Extract samples (100 µl) were added to SpinBind columns (FMC BioProducts) and were purified by using the manufacturer's protocol. Briefly, DNA was bound to a silica support under chaotropic conditions, rinsed with Tris-buffered ethanol, and eluted with 100 µl of TE buffer.

(ii) Method ii. Subsamples were subjected to agarose gel electrophoresis by loading 25 µl of crude extract onto a 1% (wt/vol) agarose gel and electrophoresing the preparation for 30 min at 5 V/cm. The ethidium bromide-stained DNA band was visualized under UV light and excised. DNA in the agarose slice was recovered by using SpinBind columns (FMC BioProducts) as recommended by the manufacturer.

(iii) Method iii. One volume of sterile distilled water and 1 volume of 7.5 M ammonium acetate were added to 100 µl of crude extract, and the mixture was incubated on ice for 5 min to precipitate the protein. The sample was centrifuged for 10 min at 10,000 × g, and the supernatant was removed and mixed with 2 volumes of ice-cold ethanol and 20 µg of glycogen. The mixture was then allowed to stand overnight at 20°C. The precipitated DNA was pelleted by centrifugation for 15 min at 10,000 × g; then it was washed with 200 µl of ice-cold 70% (vol/vol) ethanol, air dried, and resuspended in 100 µl of sterile distilled water.

(iv) Method iv. Sephadex G-200 spin columns (method of Erb and Wagner-Döbler [8], as modified by Boccuzzi et al. [3]) were used for DNA purification (12). Briefly, 1 g of dry Sephadex G-200 was hydrated, washed five times in high-salt TE buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl [pH 8]), and autoclaved. The suspension was packed aseptically into 1-ml syringes plugged at the bottom with sterile aquarium filter floss by repeated centrifugation (130 × g, 10 min) inside 15-ml sterile, screw-cap plastic tubes until the final compacted volume was 1.1 ml. The packed columns were washed with 100 µl of high-salt TE buffer, centrifuged (130 × g, 10 min), and transferred to new sterile 15-ml screw-cap plastic tubes. Subsamples (100 µl) of the crude extract were loaded into the syringes, and the DNA was eluted with three consecutive 100-µl portions of high-salt TE buffer, each followed by centrifugation (130 × g, 10 min). The final volume of eluted, purified DNA was reduced to 100 µl by butanol extraction (32).

PCR inhibition assay. To determine whether PCR inhibitors were present in the purified DNA extracts, we designed an assay based on amplification of the 16S rRNA gene of Methylomonas albus BG8 (26). Equal volumes of purified DNA solutions from three or four replicate DNA purification runs were pooled and serially diluted in 10-fold increments with sterile, distilled water. A 10-µl portion of each dilution was added to a thin-walled PCR tube and amended with 0.5 ng of purified M. albus BG8 DNA in 5 µl of distilled water. The M. albus BG8 DNA was extracted and purified from cultures by using standard methods (1). Two drops of sterile, light mineral oil were then added to each tube. After the sample was heated to 80°C, 10 µl of a concentrated PCR buffer solution was added to each tube; each preparation contained (final concentrations) 50 mM KCl, 10 mM Tris buffer (pH 8.8), 1.5 mM MgCl₂, 0.1 mg of bovine serum albumin per ml, 0.05% (vol/vol) Tween 20, 0.5 µM oligonucleotide PCR primer 10  (5), 0.5 µM oligonucleotide PCR primer P5 (11), each deoxynucleoside triphosphate at a concentration of 0.5 mM, and 0.025 U of Taq polymerase per µl. PCR amplification was carried out with a thermal cycler (model PTC-150; MJ Research, Watertown, Mass.) by using the following program: 95°C for 2 min, 30 cycles consisting of 95°C for 1 min, 60°C for 1 min, and 72°C for 2 min, and a final extension step consisting of 72°C for 5 min. After the amplification program was completed, 10 µl from each PCR tube was electrophoresed along with -HindIII DNA standards (Gibco/BRL) on a 1.5% (wt/vol) agarose gel at 4 V/cm for 1 to 2 h. The resulting 215-bp PCR product (M. albus BG8 amplicon) was visualized by staining with ethidium bromide. A PCR was considered positive if the expected 215-bp amplicon was detected. When the BG8 amplicon was detected at the same dilution of all three replicate soil or sediment samples, a score of 3 was assigned; likewise, scores of 1 and 2 were assigned to reflect successful amplification from one and two of the samples, respectively.

Influence of bead mill homogenization parameters on DNA extraction. We examined the influence of bead mill homogenization speed on the DNA yield and fragment size by using the forest soil and two different bead mill homogenizers, the Mini Bead Beater (see above) and a Mini Bead Beater-8 (BioSpec Products). The homogenization speeds used for the Mini Bead Beater were calibrated between 1,600 and 5,000 rpm by using a stroboscope to measure the beating speed. For the trials, the homogenization time was constant (5 min) and extraction procedure 1 (Table 2) with SpinBind purification (as described above) was employed. The DNA yield was quantified as described below. The DNA fragment size distribution was determined by electrophoresis (3 V/cm for 1.5 h) on a 0.8% (wt/vol) agarose gel; the standards used were the -HindIII DNA standards described above.

DNA quantification. The DNA in crude and purified extracts was quantified by comparing the fluorescence intensities of ethidium bromide-stained DNA sample bands to the fluorescence intensities of DNA standards on agarose gels by using the method of Zhou et al. (44). This method was modified by performing RNase digestion of the DNA extracts prior to electrophoresis and by including on the agarose gels negative control lanes containing only the loading dye in order to control for horizontal and vertical differences in the observed background densities of the stained gels (26). The DNA band densities on a gel (minus the background value) were measured by using an inverted, scanned, electronically printed image of the gel (42) and the public domain NIH Image program, which was developed at the U. S. National Institutes of Health (40a). A DNA standard curve was obtained for each agarose gel by using the densities (minus the background value) of the five or six smallest DNA fragments in four to six replicate lanes containing 100 to 250 ng of HindIII-digested  DNA fragments (Gibco/BRL).

Replication and statistical analyses. All statistical analyses were performed by using Minitab statistical software (Minitab Inc., State College, Pa.). Three or four replicates per DNA extraction method were used for comparative statistical analyses of the nine DNA extraction procedures. The DNA yields were normalized to the yield obtained with extraction procedure 1 for each soil and sediment by dividing the DNA yield obtained with the procedure in question by the DNA yield obtained with procedure 1. For pairwise comparisons of the procedures, two-sample, one-sided t tests were used to determine if one procedure yielded significantly more DNA than another method. To determine the efficiency of DNA recovery after purification, four replicates were used for each purification method for each soil sample type. The percentage of DNA recovered for each sample and procedure was determined by comparing the DNA yield after purification to the DNA yield obtained with no purification.

Optimized DNA extraction-homogenization-purification protocol. Quadruplicate 100-mg aliquots of freeze-dried agricultural soil, forest soil, or wetland sediment were added to a series of 2-ml screw-cap plastic vials containing 2 g of sterile, 0.1-mm-diameter zirconium-silica beads. Equal volumes (300 µl) of 100 mM NaH₂PO₄ (pH 8), an SDS lysis mixture (100 mM NaCl, 500 mM Tris [pH 8], 10% [wt/vol] SDS), and chloroform-isoamyl alcohol (24:1) were then added to each of the vials (DNA extraction procedure 3). Bead mill homogenization (2 min at 3,300 rpm with the Mini Bead Beater) was then used to physically disrupt the bacterial cells, and the crude DNA extract was recovered from the vials as described above. After the crude extract volume was reduced to 100 µl, the DNA was purified by using Sephadex G-200 columns and was quantified (see above).

	RESULTS

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Comparison of extraction procedures. When the nine different DNA extraction procedures (Table 2) were used with freeze-dried samples, all of the procedures except procedures 6 and 9 yielded ethiduim bromide-stained bands in agarose gels (Fig. 1). Although GTC extraction is useful for RNA extraction from soils and sediments (19, 40), it is clearly not suitable for DNA extraction. The size distribution of the DNA fragments (16 to 3.5 kb) obtained with each of the remaining extraction procedures (procedures 1 through 5, 7, and 8) by gel electrophoresis revealed that some limited shearing took place during extraction regardless of the physical disruption method employed. The DNA yields varied considerably from sample to sample and with the extraction procedure used (Table 3). Generally, the DNA yields were greatest with the wetland sediment samples (range, 6 to 53 µg/g [dry weight]), intermediate with the forest soil (4 to 35 µg/g [dry weight]), and smallest with the agricultural soil (1.5 to 7.9 µg/g [dry weight]). For each sample, procedures that included bead mill homogenization and an organic solvent (either phenol or chloroform) along with SDS yielded larger amounts of DNA than procedures that included three freeze-thaw cycles with SDS, enzymatic pretreatment, and the chelator Chelex 100. 

View larger version (70K): [in this window] [in a new window]   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 -HindIII marker.

Cellular lysis. The effects of the DNA extraction procedures on cell lysis were examined only with the wetland sediment. The extent of lysis varied between 63 and 81% (Table 3). All of the procedures, even the GTC treatments from which no DNA was recovered, resulted in considerable cellular lysis. ANOVA indicated that the DNA extraction procedure also had a significant effect on cellular lysis (P = 0.003). Two-sample, one-sided t tests confirmed that extraction with an organic solvent (phenol or chloroform) produced significantly greater lysis than extraction without an organic solvent (P < 0.001). Although bead mill homogenization resulted in greater lysis than three freeze-thaw cycles (68% ± 4% versus 63% ± 6%), the effect was not statistically significant (P = 0.17) and did not account for the significantly greater DNA yield. Rather, the vigorous shaking during homogenization probably resulted in the liberation of more DNA from lysed organisms into the extraction mixture instead of the DNA remaining associated with soil particles and cell debris. The results of cellular lysis in the presence of Chelex 100 (procedure 4) were also not statistically different from the results of extraction with no Chelex 100 (procedure 1) (P = 0.48). As expected, there was a strong positive correlation between DNA yield and extent of cellular lysis (r = 0.71) in for the procedures that yielded significant amounts of DNA (procedures 1 through 5, 7, and 8).

Comparison of DNA purification methods. DNA extraction procedure 4 (SDS-Chelex 100 chemical treatment combined with bead mill homogenization) resulted in the darkest DNA-containing extracts. These extracts were also found to be the most inhibited extracts in the PCR inhibition assay. Indeed, compared to all other DNA extraction procedures, procedure 4 required at least one additional 10-fold dilution to successfully PCR amplify DNA (data not shown). Thus, procedure 4 was used for subsequent tests involving four different DNA purification procedures (Table 5). In these tests the level of DNA recovery was expressed as a percentage of the DNA in the crude extract. The values obtained with the four purification methods were very different (Table 5). ANOVA demonstrated that both sample type and purification method had a significant effect on DNA recovery (P  0.001 each). When levels of DNA recovery were compared across sample types, the Sephadex G-200 column purification method resulted in the highest levels of DNA recovery (85% ± 8%), followed by the ammonium acetate precipitation method (76% ± 8%), the SpinBind purification method (68% ± 23%), and the gel electrophoresis purification method (29% ± 17%). As determined by one-sided, two-sample t tests, the level of DNA recovery after Sephadex G-200 column purification was found to be significantly greater than the level of recovery after gel electrophoresis purification (P = 0.018). However, the statistical evidence that the Sephadex G-200 column purification method yielded more DNA than the SpinBind and ammonium acetate precipitation methods was not as strong (P = 0.18 and P = 0.13, respectively).

Optimization of bead mill homogenization parameters. The effects of two key bead mill homogenization parameters (speed and duration) on DNA extraction were examined by using the forest soil samples. The homogenization devices tested (Mini Bead Beater and Mini Bead Beater-8) were from the same manufacturer (BioSpec Products), but they differed in sample capacity, design, and mechanical specifications. The Mini Bead Beater holds a single 2-ml tube, moves with wrist action through a 1.6-cm shake distance with a soft reversal, and operates at relatively high speeds (up to 5,000 rpm). In contrast, the Mini Bead Beater-8 holds eight 2-ml tubes and moves with piston action through a 3.2-cm shake distance with a sharp reversal; it operates at relatively low speeds (up to 2,700 rpm).

View larger version (19K): [in this window] [in a new window]   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).

View larger version (22K): [in this window] [in a new window]   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).

DNA recovery with the optimized extraction-homogenization-purification protocol. The DNA yields obtained with the agricultural soil, the forest soil, and the wetland sediment when the optimized protocol (corrected for purification losses) was used were 14.7 ± 2.8, 75.6 ± 9.2, and 107.9 ± 2.1 µg/g (dry weight), respectively (Table 6). These yields corresponded to extraction efficiencies of 7.7, 17.6, and 9.8 fg of DNA per bacterial cell, respectively, based on the acridine orange fluorescence microscopic counts shown in Table 1. The levels of DNA recovery from the agricultural soil, forest soil, and wetland sediment, based on an assumed average value of 9 fg of DNA per cell (38), were 86, 195, and 109%, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Selection of optimal DNA extraction and purification procedures. Data in Tables 3 through 5 and Fig. 1 through 3 provide criteria for choosing procedures and conditions in order to achieve specific DNA extraction goals. The experimental setup which was used allowed us to statistically evaluate the efficacy of individual elements of DNA extraction and purification procedures, including incorporation of chelators and organic solvents into the lysis mixtures. Our results support the results of previous studies (20, 21, 27, 33) in which higher DNA yields were obtained with bead mill homogenization than with freeze-thaw physical lysis. However, we found that including lysozyme pretreatment, Chelex 100, or GTC had detrimental effects and either decreased the DNA yield (lysozyme and GTC) or increased humic acid recovery (Chelex 100). Including organic solvents (phenol and chloroform) substantially increased the DNA yield (19 and 35%, respectively).

Selection of optimal bead mill homogenization conditions. A major effort was made in this study to optimize bead mill homogenization parameters (speed and duration) in order to obtain large quantities of high-molecular-weight DNA. Previous work has shown that bead mill homogenization may yield either highly sheared DNA or high-molecular-weight DNA (21, 22), and we were interested in reconciling these contradictory findings. We found that shearing occurred during longer homogenization times and at higher speeds. Furthermore, including chloroform in the extraction mixture, particularly when the Mini Bead Beater-8 was employed, also enhanced shearing through an unknown mechanism. Ample quantities of high-molecular-weight DNA, however, could be obtained by using either bead mill homogenizer with or without chloroform by closely monitoring bead mill homogenization speed and duration.

Evaluation of the optimized extraction-homogenization-purification protocol. An estimate of the overall efficiency of obtaining whole-community DNA is one of the most valuable criteria for assessing an extraction technique. Two methods, recovery of DNA from microorganisms added to soil samples and recovery of DNA from a known quantity of indigenous microorganisms, have been used to assess extraction efficiency. We avoided the former method for assessing recovery of DNA from soil microbes for two reasons. Added organisms do not represent the full diversity of indigenous microorganisms (large and small organisms with different capacities for lysis), and the use of such organisms does not account for soil matrix effects (partial to full protection of bacteria associated with the surfaces or interiors of soil particles) that limit cell lysis. We felt that the latter method, comparing DNA recovery to direct counts of indigenous microorganisms by using a DNA-binding fluorochrome, such as DAPI, provided a better comparison of DNA methods. DNA yields and fluorescent direct counts of bacterial cells are only occasionally reported, and the range of reported values is 0.5 to 5.36 fg of DNA per bacterial cell (8, 9, 27, 35, 44). This is equivalent to 6 to 60% DNA recovery based on an assumed average value of 9 fg of DNA per cell (38). When our optimized extraction, homogenization, and purification protocol with the Mini Bead Beater was applied to the agricultural soil, the forest soil, and the wetland sediment, the DNA recovery efficiencies were 86, 195, and 109%, respectively. These high values underscore the effectiveness of the optimized DNA extraction procedure used for freeze-dried samples. These results also show that it is necessary to assess nonbacterial DNA pools. There are several possible explanations for extraction efficiencies greater than 100%. A large number of eukaryotic microorganisms or substantial amounts of high-molecular-weight plant and animal detrital DNA could account for DNA extraction efficiencies greater than 100%. In addition, DNA extraction efficiencies are based on fluorescent direct counts of bacteria in freeze-dried soils and sediments. Initial cell lysis during the freeze-drying process could lead to an incomplete census of microorganisms and falsely inflate DNA extraction efficiencies. We found that DNA yields decreased rapidly for refrigerated samples and decreased slowly over several weeks for frozen samples and that freeze-drying a sample minimized DNA loss. Future work should explore the benefits of the freeze-drying process, which include sample preservation, initial cell lysis, and increased lysis efficiency due to cell wall damage during the freeze-drying process.