Patent Application
20230348889
Implementations relate to methods of extracting deoxyribonucleic acid from soil. Particular implementations involve scalable methods of extracting microbial DNA from small to moderately sized soil samples.
Genomic DNA from microbial populations present in soil can be extracted and analyzed to acquire species-specific data reflecting a variety of environmental processes. Various approaches to extracting DNA from soil samples have been developed, most of which utilize commercial kits containing proprietary extraction devices and reagents. While such kits can be used to generate large amounts of DNA, large yields are not necessarily desirable for certain applications, the DNA concentrations may be low, and the kit components and corresponding protocols are usually not amenable to user-specific adjustments. As such, the kits may not be scalable and may frequently generate large amounts of wasted products. The kits can also be labor intensive, thus requiring significant time and effort to extract DNA, and protocols developed for other sample types, e.g., plant or human tissue, are not compatible with soil-based DNA extraction techniques. New approaches to extracting microbial DNA from soil samples are thus desired.
Implementations provide methods of extracting genomic DNA from soil. Specific embodiments involve methods and reagents for extracting genomic DNA from microbial populations included in small to moderately sized soil samples. The methods may be readily scaled according to user preferences, and may be implemented using standard laboratory equipment and common laboratory reagents without the need for commercial kits. Example methods may involve lysing microbial cells contained in a soil-water slurry to release DNA therefrom, binding the released DNA using a silica binding solution, washing non-DNA debris from the bound DNA to isolate it, and resuspending the isolated DNA for continued analysis. The final DNA samples may be free or substantially free of humic acid and thus highly pure, thereby improving the effectiveness of PCR-based amplification and analysis.
In accordance with principles of the present disclosure, a method of extracting DNA from a soil sample may involve lysing microbial cells within the soil sample by mixing the soil sample with a sonication buffer, the sonication buffer comprising cTAB (cetrimonium bromide). The method may also involve binding DNA released from the microbial cells to a silica substrate, washing non-DNA debris from the silica substrate, isolating the DNA to the silica substrate, and eluting the bound DNA using an elution buffer.
In some examples, mixing the soil sample with a sonication buffer may be performed in an 8-mL well of a multi-well plate. In some embodiments, the soil sample may include between about 250 and about 750 μg of soil, inclusive. In some examples, the soil sample may include a mixture of soil and water, e.g., a slurry. In some embodiments, about 2 mL of the mixture of soil and water may be mixed with the sonication buffer. In some examples, the silica substrate includes a solution of silica powder and water. In some embodiments, the silica powder and water may be present in the solution at a ratio of 1:1 or about 1:1. In some examples, binding DNA released from the microbial cells to a silica substrate may involve admixing the DNA released from the microbial cells with a mixture of 200 μL (or about 200 μL) of the solution and 1 mL (or about 1 mL) of 6M guanidine thiocyanate.
In some examples, the method can further involve centrifuging the soil sample and the sonication buffer together at about 4816×g (4700 rpm) for about 5 minutes, thereby forming a supernatant containing the DNA released from the microbial cells. In some examples, lysing the microbial cells within the soil sample further involves sonicating the soil sample mixed with the sonication buffer. In some embodiments, washing non-DNA debris from the silica substrate involves resuspending and centrifuging the silica substrate in a wash buffer two or more times. In some embodiments, the sonication buffer may include about 2% w/v cTAB.
In accordance with principles of the present disclosure, a method of extracting microbial DNA from a plurality of soil samples can involve adding 2 mL (or about 2 mL) of each of the plurality of soil samples into separate wells within a multi-well plate and lysing microbial cells within each of the plurality of soil samples by mixing each soil sample with a sonication buffer, the sonication buffer comprising cTAB. The method may further involve binding the microbial DNA released from the microbial cells to silica particles suspended in a silica solution, washing non-DNA debris from the silica particles, and eluting the DNA from the silica particles to obtain isolated DNA.
In some examples, each of the separate wells within the multi-well plate holds 8 mL of liquid (or about 8 mL). In some embodiments, the multi-well plate includes 24 wells (or about 24 wells). In some examples, the sonication buffer comprises NaCl, PVP, TE buffer, RNase A, and about 2% w/v cTAB. In some embodiments, each of the plurality of soil samples comprises a 1:1 mixture of soil and water (or about 1:1). In some examples, each of the plurality of soil samples comprises about 500 μg of soil (or about 500 μg). In some embodiments, binding the microbial DNA released from the microbial cells to silica particles comprises admixing the microbial DNA with a mixture of 200 μL of the silica solution and 1 mL of 6M guanidine thiocyanate. In some examples, the silica solution includes a 1:1 mixture of silica particles and water (or about 1:1).
The soil-based DNA extraction methods provided herein can advantageously utilize a smaller amount of soil compared to preexisting methods and may reduce, minimize or eliminate humic acid content within the soil samples during extraction, thereby improving final DNA purity. The methods may also be scaled as desired to meet user preferences, making the methods adaptable to a variety of different applications, e.g., research and industrial. The final genomic DNA products may be highly concentrated and pure. The equipment required to implement the methods may be standard equipment typically found in most research or production laboratories. Specialized devices, such as the disposable bead tubes, membranes and spin columns frequently included in commercial kits, are not required to perform the methods herein, thereby providing additional benefits related to cost expenditures, accessibility and waste.
Extraction methods provided herein may involve multiple stages, which may generally include: lysis, DNA binding, washing and elution stages, with each stage comprising a plurality of steps. Methods may also involve a pre-processing stage. The lysis stage breaks apart the microbial cells included within the soil samples, thereby exposing the internal cellular components, including total genomic DNA. The DNA binding stage causes DNA molecules released from the lysed cells to bind with silica particles included in a DNA binding solution. The washing stage removes extraneous, i.e., non-DNA, soil debris, residual chemicals required in DNA extraction, and cellular components, including lipids, proteins and organelles, etc. The elution stage resuspends the isolated DNA in an elution buffer for quantification and follow-up analysis.
Reagents: Prior to performing one or more of the aforementioned stages, one or more of the reagents utilized in the extraction process may be prepared and optionally stored. Reagents utilized according to the methods herein may include NaCl, Tris-EDTA, sodium acetate, a wash buffer, a DNA binding buffer, a sonication buffer, a DNA elution buffer, and a DNA binding solution. The specific volumes, concentrations and/or pH levels provided herein for each reagent may be for example purposes only, and should not be viewed as limiting. For example, the amount and/or properties of one or more reagents may be modified according to user preferences such that the disclosed methods are fully scalable. In some embodiments, for example, the volumes of one or more stock reagents may be multiplied to accommodate more plates utilized for an extraction. Stock solutions having higher concentrations than those disclosed below, for example, may be prepared and diluted upon use. In addition, the total volume of each reagent can be readily adjusted depending on the number of soil samples simultaneously used for DNA extraction.
In some examples, the NaCl (sodium chloride) may be 5M (or about 5M), having a molecular weight of 58.44 g/mol. To prepare 5M NaCl, 146.1 grams of NaCl (or about 146.1 grams) may be mixed with distilled water to reach a total volume of 500 mL (or about 500 mL).
In various examples, 10X Tris-EDTA (ethylenediamine tetraacetic acid or “TE”) (or about 10Δ) may be prepared by mixing 100 mL of 1M Tris-HCl, pH 8.0 (or about 100 mL) with 20 mL of 0.5M EDTA, pH 8.0 (or about 20 mL of about 0.5M EDTA) and adding distilled water to reach a total volume of 1 L (or about 1 L).
The sodium acetate may be 3M (or about 3M), having a molecular weight of 82.03 g/mol. To prepare the sodium acetate, 123.05 grams of sodium acetate (or about 123.05 grams) can be mixed with water to reach a total volume of 500 mL (or about 500 mL). The pH may then be adjusted to 5.2 (or about 5.2), for example using glacial acetic acid.
The wash buffer can include 0.05M NaCl (or about 0.05M) and may comprise 90% ethanol (or about 90%). The wash buffer may be prepared by mixing 3 mL of 5M NaCl (or about 3 mL of about 5M NaCl) with 270 mL of 100% ethanol (or about 270 mL of about 100% ethanol) and adding distilled water to reach a total volume of 300 mL (or about 300 mL).
The DNA binding buffer may comprise 6M guanidine thiocyanate (or about 6M), having a molecular weight of 118.2 g/mol. To prepare the DNA binding buffer, 70.92 grams of the 6M guanidine thiocyanate (or about 70.92 grams of about 6M guanidine thiocyanate) can be mixed with distilled water, reaching a total volume of 100 mL (or about 100 mL).
The sonication buffer can comprise 0.5M NaCl (or about 0.5M), 3% PVP (or about 3%) (polyvinylpyrrolidone, weight 40,000), 10×TE (or about 10×), RNase A, and cTAB (cetrimonium bromide), and it may be prepared by mixing 25 mL of 5M NaCl (or about 25 mL of about 5M NaCl) with 7.5 grams of PVP (or about 7.5 grams), and 2% w/v cTAB (or about 2% w/v), and adding 10×TE (or about 10×) up to a total volume of 250 mL (or about 250 mL). The RNase A may be added just prior to use of the sonication buffer. In embodiments, about 50 μL of RNase A at a concentration of 5 μg/μL may be added per 50 mL of sonication buffer. The sonication buffer may specifically exclude additional reagents, including proteinase K, SDS (sodium dodecyl sulfate), and various enzymes, e.g., lysozymes and glusulase, thereby further simplifying the processes described herein.
The DNA elution buffer can comprise 0.1×Tris-EDTA (or about 0.1×), which may be prepared by mixing 2 mL of 10×Tris-EDTA (or about 2mL of about 10×Tris-EDTA) with 48 mL of distilled water (or about 48 mL).
The DNA binding solution utilized herein, which may include a combination of silica powder and water, may replace the commercial binding solutions and associated devices commonly included in commercial kits. Preparation of the DNA binding solution can involve washing and suspending silica powder with water one or more times, e.g., 2, 3, 4 or more times. In one embodiment, about 800 mg of silica powder, e.g., CELITE® 545, can be poured into a 50 mL centrifugation tube, e.g., a BD FALCON™ tube, such that the powder reaches to about the 2.5 mL mark on the tube. About 30 mL of water, which may be distilled, can be added to the tube and mixed with the silica powder by vigorously shaking and/or vortexing. The resulting slurry may then be allowed to settle for approximately 15 minutes, or at least until the powder and water separate. The water can then be decanted and another aliquot of about 30 mL of water added to the silica powder. The aforementioned washing steps, i.e., mixing, separating and decanting, can then be repeated two or more times. After the final washing step, the silica powder can be resuspended in a fresh aliquot of water. The volume of water used to resuspend the silica may vary. In some examples, the volume of water may be approximately equal to the volume of silica, such that the ratio of silica to water is about 1:1 and the resuspension fills about 5 mL of the centrifuge tube. The finished DNA binding solution can be stored at about room temperature, e.g., 20-25° C., until further use. The DNA binding solution may comprise silica and water, only, without any additional components, such as potassium iodide. The simplified solution may provide yet another advantage over preexisting systems.
Immediately prior to use, the DNA binding solution can be resuspended by manually shaking, pipetting and/or vortexing. About 200 μL of the DNA binding solution can then be transferred, e.g., pipetted, into each of one or more wells defined by a multi-well plate. The capacity of each well can be 8 mL in some embodiments, and the number of wells included in the plate may be 24, although the number of wells is not critical. For example, the capacity of each well in a 96-well plate can be 2 mL. Other wells within a multi-well plate may have a 10 mL capacity. The number of wells needed, whether in one plate or multiple, depends on the number of soil samples evaluated, as each soil sample is assigned to one well. Because the methods disclosed herein do not rely on predefined kits, which can only be used to process a limited number of samples simultaneously, the number of soil samples can be scaled up or down as desired by a user, limited only by the number of multi-well plates available and/or the number of plates that can fit in a centrifuge. For example, DNA from about 84 separate soil samples can be simultaneously extracted according the methods herein, such that about 500 samples can be reasonably extracted by one user in one day. This marks a significant improvement over preexisting methods, which may be limited to about 25 samples per user per day. In addition, the multi-well plates can be washed and reused. For instance, plates may be re-washed after each extraction.
During the transfer of DNA binding solution to each well, the remaining stock solution may be periodically agitated to maintain uniform distribution of the silica in the suspension. To each well containing DNA binding solution, 1 mL of the DNA binding buffer can then be added and used to resuspend the silica. Resuspension can be achieved by pipetting (repeatedly up and down) the mixture of DNA binding solution and buffer.
Pre-processing stage: Methods herein may not be limited to a particular soil composition type, and thus may be amenable to soils of varying moisture levels, including soil with various levels of sand, silt, clay, peat, organic matter, etc. One or more soil pre-processing steps may be implemented before the lysis stage. For example, unlike preexisting approaches that may utilize dry soil, methods herein may utilize slurries of soil and water. As such, a pre-processing step can involve wet-milling the soil sample(s) used for DNA extraction. Wet-milling the soil may involve mixing, e.g., blending, the soil with water to form a slurry. Replicates of each soil sample can be prepared, along with one or more control samples. The amount of soil used to prepare each slurry sample may vary, and may be less than amounts required for preexisting DNA extraction protocols, which may recommend about 10 grams of soil per sample. In various embodiments, the amount of soil used to create a slurry for each sample can range from about 250 mg to about 1 gram, about 350 mg to about 750 mg, about 450 mg to about 550 mg, about 475 mg to about 525 mg, or about 500 mg. In some examples, dried, ground soil may still be used. Such examples may be performed with some variations, e.g., without the use of silica as a binding agent.
Lysis Stage: The lysis stage bursts, punctures and otherwise breaks apart microbial cells present within the soil samples, thereby exposing the internal cellular components, including total genomic DNA, organelles, proteins, etc. During the lysis stage, samples of each slurry, for example about 2 mL, can be pipetted into each well of the multi-well plate(s). To each well, 2.5 mL of sonication buffer can then be added and the plate(s) sonicated for about 30 seconds to about 2 minutes to lyse the cells within the slurry. In some embodiments, RNase A may be added to the sonication buffer just prior to sonication. For example, about 50 μL of RNase A at a concentration of 5 μg/μL may be added per 50 mL of sonication buffer, such that 12.5 μg of RNase A are added to each well upon addition of 2.5 mL sonication buffer. The sonication settings applied to the samples may vary. In one example, the plate(s) can be sonicated using a 117V VWR® Ultrasonic Cleaner. The inclusion of cTAB within the sonication buffer, for example at about 2% w/v, can precipitate humic acid and humic acid aggregate components during sonication, facilitating the removal of such substances, which may otherwise contaminate the isolated DNA and inhibit downstream analysis, e.g., PCR. After sonication, 1 mL of 3M sodium acetate can be added to each well, followed by optional mixing, and each plate placed in freezer set at −20° C. for 10 minutes. The plates may then be removed from the freezer and centrifuged at 4816×g (4700 rpm) for 5 minutes at room temperature, thereby separating the released cellular components and DNA (supernatant) from the solid soil debris. The lysis stage may be performed without the use of any metal beads and/or grinding.
DNA Binding Stage: The DNA binding stage causes DNA molecules released from the lysed cells to bind with DNA-binding particles, such as the silica particles included in the DNA binding solution. The DNA binding stage may first involve preparing 1.2 mL (or about 1.2 mL) of a fresh mixture of DNA binding solution and DNA binding buffer for each soil sample, which can be added to each 8-mL well in a clean multi-well plate. Each aliquot of the fresh mixture can comprise 200 μL (or about 200 μL) DNA binding solution and 1 mL (or about 1 mL) of DNA binding buffer. The supernatant generated during centrifugation of the lysed cells can then be added to each well containing the 1.2-mL aliquot of DNA binding mixture. For each sample, the volume of supernatant removed after centrifugation and combined with DNA binding mixture may be about 4 mL. Care should be used to ensure that no soil is transferred with the supernatant. Each plate may then be covered with a paraffin film, e.g., PARAFILM®, and stacked using metal stacking plates, if necessary. Stacked plates may be connected, e.g., via clamps. The covered plates can then be incubated for about 15 minutes at room temperature on a shaker set at 200 rpm. After 15 minutes, the paraffin film can be removed, and the plates centrifuged at or about 4816×g (4700 rpm) for 5 minutes at room temperature, thereby forming a pellet comprised of DNA-bound silica and a liquid supernatant in each well. The supernatant can be removed, e.g., using a pipette, such that only the DNA-bound silica pellet remains in each well. Non-DNA components that remain associated with the silica can be removed during the washing stage.
Washing Stage: The washing stage removes extraneous, i.e., non-DNA, soil debris, residual extraction chemicals, and cellular components, including proteins, organelles, lipids, membranes, etc., from the silica particles. The washing stage may involve adding 1 mL of wash buffer to each well containing a DNA-bound silica pellet and resuspending the pellet therein. Resuspending the pellet components can involve gently shaking each plate against a table, for example. The resuspension can then be centrifuged at or about 1962×g (3000 rpm) for 1 minute at room temperature. The wash buffer supernatant generated via centrifugation can be removed by pipetting, and the washing step can be repeated using a fresh aliquot of 1 mL wash buffer. After a second round of centrifugation, the supernatant can again be removed via pipette. To remove as much supernatant as possible, the plates can be tipped at an angle, thereby pooling any residual liquid in each well for pipette removal. The DNA-bound silica pellets can be washed 2 or more times, e.g., 3, 4 or 5 times. After the final washing step, the DNA-bound silica can be allowed to dry in a vacuum hood for up to about 1 hour at room temperature.
Elution Stage: The elution stage elutes the isolated DNA in a buffer, such as the elution buffer described herein. The elution stage can involve adding 300 μL (or about 300 μL) of the DNA elution buffer to each well containing dried silica, and optionally pipetting to mix. The mixture of DNA elution buffer and silica can then be incubated for about 20 minutes at room temperature under gentle agitation, for example by placing the samples on a shaker set at or about 200 rpm. The plates should be kept facing up to keep the silica positioned at or near the bottom of each well. After incubation, the plates can be centrifuged at or about 4816×g (4700 rpm) for 5 minutes at room temperature to pellet the silica, thereby separating the silica from the DNA contained in the elution buffer. About 200 μL of the supernatant can then be removed from each well and placed in a fresh well. Care should be taken to avoid transferring any of the silica with the supernatant, which contains the isolated DNA.
One or more post-extraction steps may then be implemented to analyze the quantity, quality and/or identity of the isolated DNA. For example, each DNA sample can be quantified using a DNA quantification plate reader, e.g., a SPECTROSTAR® Nano reader sold by BMG Labtech. The DNA can be stored at −20° C. for long-term storage or 4° C. for shorter term storage.
In the example shown, the method 100 begins at block 102 by “lysing microbial cells within a soil sample by mixing the soil sample with a sonication buffer, the sonication buffer comprising cTAB (cetrimonium bromide).” The method 100 continues at block 104 by “binding DNA released from the microbial cells to a silica substrate.” The method 100 continues at block 106 by “washing non-DNA debris from the silica substrate.” The method 100 continues at block 108 by “isolating the DNA from the silica substrate.” The method continues at block 110 by “eluting the isolated DNA in an elution buffer.”
In the example shown, the method 200 begins at block 202 by “adding 2 mL of each of a plurality of soil samples into separate wells within a multi-well plate.” The method 200 continues at block 204 by “lysing microbial cells within each of the plurality of soil samples by mixing each soil sample with a sonication buffer, the sonication buffer comprising cTAB.” The method continues at block 206 by “binding the microbial DNA released from the microbial cells to silica particles suspended in a silica solution.” The method continues at block 208 by “washing non-DNA debris from the silica particles.” The method continues at block 210 by “separating the DNA from the silica particles to obtain isolated DNA.”
The DNA yield obtained according to the methods described herein may be approximately equal to or better than the yield obtained using preexisting commercial kits. About 1 μL of each DNA sample (in the elution buffer) may be sufficient to perform PCR. All reagents may be completely used, such that no waste remains, and all plates washed and re-used. The quantity and concentration of DNA may be suitable for a vary of settings and applications. For example, research institutions, industry laboratories, soil production laboratories, government laboratories, etc. may all implement the methods herein. DNA from various microbial species can be isolated and analyzed, including but not limited to: Bacillus anthracis, Bacillus subtilis, and Streptomyces species.
As used herein, the term “about” modifying, for example, the quantity of a component in a composition, concentration, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or components used to carry out the methods, and like proximate considerations. The term “about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture. Where modified by the term “about” the claims appended hereto include equivalents to these quantities.
Similarly, it should be appreciated that in the foregoing description of example embodiments, various features are sometimes grouped together in a single embodiment for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various aspects. These methods of disclosure, however, are not to be interpreted as reflecting an intention that the claims require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment, and each embodiment described herein may contain more than one inventive feature.
Although the present disclosure provides references to preferred embodiments, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.
This application claims priority to U.S. application Ser. No. 16/165,847 filed Oct. 19, 2018 entitled “SOIL-BASED DNA EXTRACTION” which is incorporated by reference herein, in the entirety and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16165847 | Oct 2018 | US |
| Child | 18348543 | US |
