By: Aesha Patel and David Burden
OPS Diagnostics, LLC.
Isolating quality DNA from the soil microbiome can be challenging. The presence of humic acid makes isolating DNA difficult, as humic acid co-purifies. Chromatographic and extraction methods do not always result in clean end products. Commercial soil DNA purification kits can yield cleaner DNA than homegrown methods, but results are not always ideal and costs can be significant. For the purposes of this study, the standard Synergy™ 2.0 plant DNA isolation protocol was modified by adding a CTAB DNA precipitation step to the process, where humic acids remain in solution under favorable conditions for DNA precipitation. By adding the CTAB precipitation step, quality DNA was effectively isolated in comparable yield from agricultural soil, and at higher yield from garden soils, compost, and thatch as compared to the most popular Commercial Kit. The Synergy™ process was more effective at removing polyphenolics from the final product.
Soil microbes have been intensely studied since the isolation of antibiotic producing bacteria during the middle of the 20th century. Since the 1990s, and particularly in the past few years, interest has expanded beyond analysis of individual isolates to the microbiome. Microbiomes are examined at the molecular level by DNA analysis, an objective made possible by NextGen Sequencing, which requires the extraction of quality DNA from the soil. The isolation of DNA from soil for microbiome analysis can be challenging due to both the extreme variability of soil composition and from the inherent nature of its composition. Polyphenolic acids found in soil, such as humic acid, are a significant issue when isolating microbiome DNA.
Humic acid, which makes up a significant proportion of the organic matter of soil, is generated from the decomposition of lignin. These acids represent a heterogeneous solution of polyphenolic rings that have consistently been found to co-purify with nucleic acids (Fatima et al., 2011). Humic acids that contaminate DNA have been shown to bind protein and thus potentially interfere with downstream manipulation of the DNA, including PCR (Tsai and Olson, 1992).
Many strategies have been applied to separating humic acids from DNA during isolation. Common approaches include the use of polyvinylpolypyrrolidone (PVPP), an insoluble polymer that binds humic acid (Steffan et al., 1988), separating higher molecular weight DNA from humic acids using gel filtration (Tsai and Olson, 1992), and adsorbing DNA to chromatography matrices followed by washing away humic acid contaminants and subsequent elution (Roose-Amsaleg et al., 2001). The range of soil types makes any single method impractical, thus Lever et al. (2015) proposed a modular extraction strategy that addresses the diverse nature of soil samples. Multiple separation techniques are used in all approaches.
OPS Diagnostics’ Synergy™ chemistry originally designed for plants was tested and modified to effectively isolate DNA from soil. Synergy™ incorporates the bead beating step of homogenization into the purification scheme using a proprietary chemistry that binds contaminants to the Synergy™ grinding matrix. By modifying the existing process to include a CTAB precipitation step, humic acids were effectively removed from the DNA preparation, yielding high quality DNA when measured spectrophotometrically.
Soil Samples: Soil was collected from agricultural fields, gardens and compost up to a depth of 1 inch. Samples were collected in 50 ml conical centrifuge tubes and stored refrigerated. Thatch was collected from lawns directly under the verdure, which included soil, roots, and decaying leaves. All samples were passed through a colander to remove large particles and to yield a more homogeneous sample. Pre-treated samples were stored at 4°C.
Standard Synergy™ 2.0 Protocol: Soil (50 mg) was added to a Homogenization Tube along with 500 µl of Homogenization Buffer containing CTAB (cetyltrimethylammounium bromide), followed by bead beating at 4,000 rpm for 2 minutes in either an HT Mini™ or HT24™ homogenizer. Lysates were centrifuged at 5900 x g (8,000 rpm) for 5 minutes, and the supernatant was transferred to a microfuge tube followed by the addition of 0.7 volume isopropanol. The solution was incubated at -20°C for 20 minutes and then run through a Silica Spin Column and centrifuged at 5900 x g (8000 rpm) for 2 minutes. The column was washed twice with 250 µl of ice cold 70% ethanol and the flow through was discarded. The column was centrifuged dry for 30 seconds at 13,400 x g (12,000 rpm) to remove remaining liquid. To elute the DNA, the spin column was placed in a clean collection tube with 50 µl of molecular biology water and centrifuged at 5900 x g (8000 rpm). DNA was analyzed using a DeNovix DS11 spectrophotometer.
Modified Synergy™ 2.0 Protocol: Soil (150 mg) was added to a Homogenization Tube with 700 µl of Homogenization Buffer followed by bead beating in an HT Mini™ or HT24™ at 4000 rpm for 2 minutes. The lysate was centrifuged at 5900 x g (8000 rpm) for 5 minutes and the supernatant was transferred to a new microfuge tube. The supernatant was centrifuged again at 11,200 x g (11,000 rpm) for 30 seconds to pellet any fine particles that may have transferred during decanting. The supernatant was transferred to a new microfuge tube and two (2) volumes of low salt Dilution Buffer were added, followed by vortexing, and a 10 minute incubation at room temperature. The solution was centrifuged at 13,400 x g (12,000 rpm) for 5 minutes to pellet the DNA. The supernatant was discarded, and the pellet was dissolved in 200 µl of high salt Resuspension Buffer, followed by the addition of 200 µl of isopropanol. This solution was incubated at -20°C for 20 minutes and then transferred to a Silica Spin Column and centrifuged at 5900 x g (8000 rpm). The effluent was discarded and the column was then washed twice with 250 µl of ice cold 70% ethanol. The column was centrifuged dry for 30 seconds at 13,400 x g (12,000 rpm) to remove any remaining liquid. The spin column was placed in a clean collection tube with 50 µl of molecular biology water added, followed by centrifugation at 5900 x g (8000 rpm). DNA was analyzed using a DeNovix DS11 spectrophotometer.
Commercial Kit Protocol: The kit included all reagents and disruption tubes. The isolation involved adding 250 mg of soil to disruption tubes containing garnet and homogenization buffer (supplied) supplemented with an additional 60 µl of an SDS buffer. The resulting lysate was cleared by centrifugation at 10,000 x g for 30 seconds. The supernatant was transferred to a new tube where 250 µl of a precipitation solution was added to remove inorganic and organic impurities. After 5 minutes incubation at 4°C, the tube was centrifuged at 10,000 x g for 1 minute. Up to 600 µl of supernatant was transferred to a new tube, and 200 µl of a humic acid precipitating solution was added followed by 5 minutes incubation at 4°C. This solution was centrifuged for 1 minute at 10,000 x g, and 750 µl was transferred to a new tube. A concentrated salt solution (1.2 ml) was added and mixed thoroughly. This solution was applied to a spin column in three increments of 650 µl with 1 minute of centrifugation at 10,000 x g following each application. The spin column was washed once with 500 µl of an ethanol wash solution and then eluted with 50 µl of molecular biology grade water.
The Synergy™ 2.0 plant DNA isolation protocol was applied to soil and compared to a popular Commercial Kit as regards yield and purity. Initial testing of Synergy™ for the isolation of DNA from soil microbiome yielded less than optimal results (Table 1). Purity ratios showed significant amounts of polyphenolics and possible protein contamination. DNA eluted from spin columns retained a yellow tint characteristic of humic acids. Compared to the Commercial Kit, the standard Synergy™ protocol was less effective, and total yields were lower. Also, the initial sample mass processed using Synergy™ was 50 mg versus 150 mg for the Commercial Kit, thus total yields were lower.
| Methods | Sample Size (mg) | Total Yield (µg) | Conc. (ng/µl) | 260/230 | 260/280 |
| Synergy™ 2.0 | 50 | 5.1 | 102.12 | 0.82 | 1.42 |
| Commercial Kit | 250 | 2.5 | 48.97 | 1.54 | 1.75 |
| Modified Synergy™ 2.0 | 150 | 1.6 | 31.50 | 1.62 | 1.85 |
In the development of the modified Synergy™ protocol, humic acids consistently co-purified with DNA. Chromatographic techniques, such as ion exchange, normal phase, reverse phase, gel filtration, and mixed mode, failed to effectively separate the humic acids from DNA (data not shown). The same can also be said for precipitation techniques using alcohol.
For plants, the standard Synergy™ 2.0 protocol makes use of a reverse phase chromatography process to capture contaminants and absorption to silica to wash and concentrate DNA. The chemistry also prevents the formation of polyphenolics, which can interfere with later manipulations. The reverse is not true, however, since the basic process does not effectively separate soil polyphenolic acids from the DNA.
As standard precipitation and chromatographic methods fail to separate DNA and humic acids, either alone or in tandem, a historically popular, but currently less used, approach to purifying DNA from plants was assessed. CTAB is a very popular detergent used for plant DNA isolation procedures because it is effective in separating polysaccharides from DNA. Polysaccharides are insoluble in CTAB with high salt. If the salt concentration is lowered, however, DNA precipitates in the presence of CTAB.
The Synergy™ Homogenization Buffer contains CTAB. By lowering the salt concentration of the lysate it was hypothesized that the CTAB would precipitate the DNA, leaving the humic acids in solution. The high density and evenly spaced phosphates of the DNA backbone, when ionized, provide a point of ionic attraction for the positively charged CTAB. As the ammonium groups pair with the negatively charged phosphates, it is theorized that the hydrophobic tails of CTAB (cetyl group) aggregate and cause the DNA to precipitate. This occurs under low salt conditions, while in high salt, it is believed that the cations compete with CTAB for the phosphates and prevent DNA precipitation. Though humic acids contain hydroxides that can ionize, the random arrangement of the phenolic groups and resonance of pi electrons are thought to attract less CTAB. The result is that humic acid remains in solution while DNA precipitates.
With low salt and CTAB, DNA is pelleted and subsequently dissolved by adding salt. The DNA can be cleaned further by binding and eluting from a spin column. This modified Synergy™ 2.0 protocol, as seen in Table 1, worked in removing the humic acids from the purified DNA, leaving a cleaner product than the commercially available kit for further downstream procedures. It should be noted that the additional purification step comes at the expense of yield. The Commercial Kit had the highest total yield, though the sample size was two thirds larger. The basic Synergy™ protocol generated the highest yield of 5.1 µg from the smallest sample size, which suggests that increasing the number of purification steps negatively affects yield. This is also a common issue with protein purification schemes.
The modified Synergy™ 2.0 protocol was further compared to the Commercial Kit using different soils found in the region. Agricultural soil was obtained from different active farm fields present in the region, including soybean, corn, blueberry, and cranberry. Thatch, compost, and garden soil were also obtained locally. For all samples, protocols were run at least six times and the averages were recorded on Table 2.
| Modified Synergy™ 2.0 | Commercial Kit | |||||||
| Types of Soil | Yield (µg) | ng/mg soil | 260/230 | 260/280 | Yield (µg) | ng/mg soil | 260/230 | 260/280 |
| Agricultural | 0.87 | 5.82 | 1.95 | 1.88 | 1.10 | 4.39 | 1.36 | 1.77 |
| Thatch | 11.15 | 74.30 | 2.11 | 1.85 | 2.06 | 8.24 | 2.01 | 1.89 |
| Compost | 8.03 | 53.50 | 1.67 | 1.82 | 2.35 | 9.41 | 1.99 | 1.89 |
| Garden | 4.73 | 31.50 | 1.81 | 1.73 | 2.45 | 9.79 | 1.54 | 1.75 |
As Lever et al. (2015) show in their detailed analysis of soil purification strategies, no one soil purification method is applicable to all soil types. The comparison of the results from modified Synergy™ 2.0 protocol to the Commercial Kit protocol clearly supports this notion. With agricultural samples, the Commercial Kit produced slightly higher yields while Synergy™ produced cleaner DNA, as measured by 260/280 and 260/230 ratios. Aside from the agricultural soil results that were relatively comparable, the modified Synergy™ 2.0 protocol yielded significantly more DNA from 40% less sample (150 mg vs. 250 mg). The differences in yield may be a function of the capacity of the spin column since a large amount of lysate from the Commercial Kit is applied (in three increments) though yield is one half to one fifth that of Synergy™. The spin column capacity of the Commercial Kit appears to peak around 2.5 µg of DNA. By comparison, the capacity of a Synergy™ spin column is 45 µg of DNA.
Both processes demonstrated the ability to produce relatively clean DNA. Both systems generated 260/280 ratios relatively close to 1.8. This demonstrates that protein, RNA, and other molecules that absorb light in that region are absent. The 260/230 ratio, which is more often associated with phenolics like humic acid, was closer to the desired 2.0-2.2 ratio for all the samples using the modified Synergy™ protocol than the Commercial Kit protocol, with the exception of compost. Since Synergy™ had much higher total yields, removing the remaining contaminants, i.e., those absorbing at 230 nm, may be as simple as using less sample.
In terms of cost, each sample processed using the Commercial Kit cost in excess of $5/sample. The existing Synergy™ 2.0 Plant DNA Extraction Kit costs $2.25/sample. The modification of the existing Synergy™ kit, with the addition of two new buffers to facilitate the modification of Synergy™ 2.0 for soil microbiome work, may increase sample cost to $2.50/sample. By any comparison, the Synergy™ chemistry is a more cost effective option.