Method for extracting DNA (Deoxyribose Nucleic Acid) from marine sediments

Abstract

The invention discloses a method for extracting DNA from marine sediments, and belongs to the technical field of DNA extraction. The method comprises the following steps: adding sodium hexametaphosphate and a cell lysis solution into the marine sediment, uniformly mixing, incubating and centrifuging to obtain supernate; adding a chloroform-isoamyl alcohol mixed solution into the supernate, uniformly mixing, centrifuging, and removing an organic phase to obtain a water phase; and adding linear polyacrylamide and sodium chloride into the water phase, uniformly mixing, adding ethanol, precipitating in a dark place, and centrifuging to obtain the DNA extract. Compared with the prior art, the method provided by the invention has the advantages that the DNA in the marine sediment can be effectively extracted, the extraction amount is high, the concentration is high, and the obtained DNA can better meet the metagenome sequencing requirement and the subsequent PCR reaction requirement.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA extraction, and in particular relates to a method for extracting DNA from marine sediments. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In the study of marine microbial molecular ecology, the acquisition of high-quality DNA is the basis for studying the microbial community and its structure in the marine environment. However, due to the low content of microorganisms in deep-sea environmental samples, the adsorption of nucleic acids by clay, the degradation and loss of nucleic acids during the extraction process, the co-extraction of impurities such as enzymes and inhibitors, and the lack of purification efficiency have an adverse effect on the quality and yield of nucleic acid extraction.

[0004] Existing DNA extraction methods, such as DNA extraction kits and portable methods for soil sediments, etc., obtain DNA content that is too low to perform metagenomic sequencing (generally the minimum requirement is >200ng), or the obtained DNA has a high yield but a high impurity content, which inhibits the subsequent PCR reaction. The problems of insufficient extraction amount and low purity in the DNA extraction methods in the prior art seriously limit the in-depth development of related research and development. Therefore, there is an urgent need for a method that can effectively extract DNA, especially a technology for extracting microbial DNA in deep marine sediments. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for extracting DNA from marine sediments. The present invention extracts and purifies DNA from deep marine sediment samples, and the obtained DNA concentration and total amount can better meet the requirements of metagenomic sequencing.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A first aspect of the present invention provides a method for extracting DNA from marine sediments, comprising:

[0008] Sodium hexametaphosphate and cell lysate are added to the marine sediment, mixed, incubated, and centrifuged to obtain a supernatant;

[0009] Add chloroform-isoamyl alcohol mixture to the supernatant, mix well, centrifuge, remove the organic phase, and obtain the aqueous phase;

[0010] Linear polyacrylamide and sodium chloride were added to the aqueous phase, and ethanol was added after mixing. The mixture was precipitated in the dark and centrifuged to obtain a DNA extract.

[0011] It should be noted that the marine sediments are immediately collected from the seabed and DNA is extracted using the method of the present invention, or are frozen and stored at a temperature below -80°C, and then DNA is extracted using the method of the present invention according to actual needs.

[0012] It should be noted that the deeper the location of the general marine sediment, the lower the biomass and the more difficult it is to extract. The present invention tested sediments more than 3 meters deep, and successfully extracted >200ng of DNA from marine sediments with a depth of 3.7m. It can be seen that the method provided by the present invention is universal, can successfully extract marine sediments of different depths, and can effectively extract DNA from marine sediments with a depth of more than 3 meters, with high extraction amount and high concentration, and the obtained DNA can better meet the requirements of metagenomic sequencing and subsequent PCR reactions.

[0013] In some embodiments of the present invention, the method further comprises adding grinding balls to the marine sediments. The grinding balls are added to shake the marine sediments, sodium hexametaphosphate and cell lysate to make them more fully mixed. Cells in the marine sediments are lysed and proteins are denatured by mechanical and chemical methods.

[0014] It is understandable that the mixing and incubation time can be extended according to actual conditions to ensure continuous mixing of the sample.

[0015] Preferably, the grinding balls are zirconium beads, the particle size of the zirconium beads is 1.8-2.2 mm, and the volume of the zirconium beads is 10%-20% of the volume of the device used.

[0016] In some embodiments of the present invention, the ratio of sodium hexametaphosphate, cell lysate and marine sediment is 0.8-1.2 nmol: 0.4-0.6 mL: 0.2-0.3 g;

[0017] The sodium hexametaphosphate is prepared into a solution of 8-12 mM and then added into the marine sediment;

[0018] The cell lysis solution comprises: 28-32 mM Tris-HCl, 28-32 mM disodium EDTA, 1% Triton X-100, 790-810 mM guanidine hydrochloride, and a pH of 9.8-10.2.

[0019] The present invention adds sodium hexametaphosphate to the marine sediments. The adsorption characteristics of sodium hexametaphosphate are similar to those of nucleic acids. Sodium hexametaphosphate is coated on the surface of marine sediments to reduce nucleic acid adsorption and greatly improve the efficiency of recovering nucleic acids from mineral-rich and nutrient-poor marine sediments.

[0020] In some embodiments of the present invention, the composition of the cell lysis solution is: 30 mM Tris-HCl, 30 mM disodium EDTA, 1% Triton X-100, 800 mM guanidine hydrochloride, pH 10.0.

[0021] In some embodiments of the present invention, an equal volume of chloroform-isoamyl alcohol mixture is added to the supernatant. It is understood that the equal volume means that the amount of chloroform-isoamyl alcohol mixture added is the same as the volume of the supernatant. For example, when the volume of the supernatant is 1 mL, the amount of chloroform-isoamyl alcohol mixture added is 1 mL.

[0022] Preferably, an equal volume of a chloroform-isoamyl alcohol mixture is added to the supernatant, and after mixing and centrifugation, if the aqueous phase is clear and free of chloroform or particles, the obtained aqueous phase is subjected to the next step of treatment; otherwise, the steps of adding an equal volume of a chloroform-isoamyl alcohol mixture to the obtained aqueous phase and mixing and centrifuging are repeated until a clear aqueous phase free of chloroform or particles is obtained to avoid the transfer of chloroform and sediment particles.

[0023] Chloroform removes residual proteins, membrane lipids, and detergents by dissolving or gathering at the aqueous phase interface. Isoamyl alcohol helps to form a clear aqueous-chloroform interface. If the aqueous-oil interface breaks down during this step, centrifuge again for 1 minute.

[0024] It should be noted that after mixing and centrifugation, the water phase is on the top and the oil phase is on the bottom. A pipette can be used to directly transfer the water phase and avoid introducing oil phase impurities into the water phase.

[0025] In some embodiments of the present invention, in the chloroform-isoamyl alcohol mixture, the volume ratio of chloroform to isoamyl alcohol is 22-26:1, preferably 24:1.

[0026] In some embodiments of the present invention, the concentration of the linear polyacrylamide (LPA) in the aqueous phase is 18-22 μg / mL, preferably 20 μg / mL.

[0027] In some embodiments of the present invention, the sodium chloride is added to the aqueous phase in the form of a 4.8-5.2M sodium chloride solution, and the amount of the sodium chloride solution added is 0.15-0.25 times the volume of the aqueous phase;

[0028] The amount of ethanol added is 2-3 times, preferably 2.5 times, of the total volume of the aqueous phase and the sodium chloride solution; preferably, the ethanol is anhydrous ethanol. It should be noted that after the anhydrous ethanol is added, the mixed solution contains >70% ethanol.

[0029] DNA precipitation is a key step in concentrating and purifying extracted DNA. The present invention sequentially adds LPA, sodium chloride and ethanol to the aqueous phase, and sodium chloride binds to the phosphate backbone of DNA, causing it to precipitate in the presence of >70% ethanol. LPA, as a co-precipitant, can improve the efficiency of DNA precipitation and promote the formation of precipitates. In the present invention, the order of adding LPA and ethanol is very important. If ethanol is added first, LPA and ethanol are mixed in advance, resulting in LPA precipitating before contacting DNA and failing to play a corresponding role.

[0030] In some embodiments of the present invention, the method further comprises the step of purifying the DNA extract;

[0031] Wherein, the purification includes purifying the obtained DNA extract using a purification kit.

[0032] In some embodiments of the present invention, the purified DNA extract is stored at -20°C to -80°C. The purified DNA extract can be stored for a long time at -20°C to -80°C.

[0033] In some embodiments of the invention, the purification kit comprises an EZNA Soil DNA Kit.

[0034] It should be noted that the obtained DNA extract can be purified completely according to the instructions of the EZNA Soil DNA Kit, and some steps can be appropriately adjusted according to actual conditions.

[0035] The beneficial effects of the present invention are:

[0036] The present invention selects different reagents to extract DNA from marine sediments, effectively extracts DNA from marine sediments, has a high extraction amount and a high concentration, and the obtained DNA can better meet the requirements of metagenomic sequencing and subsequent PCR reactions, thereby solving the problems in the prior art that the extracted DNA has a low content and cannot be used for metagenomic sequencing, and that high impurities inhibit subsequent PCR reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 The DNA AATI detection peak diagram (Agilent 5400) (A) and simulated gel diagram (B) of the DNA product obtained in Example 1 of the present invention;

[0039] Figure 2The DNA AATI detection peak diagram (Agilent5400) (A) and simulated gel diagram (B) of the DNA product obtained using the OMEGA EZNA Soil DNA Kit;

[0040] Figure 3 The DNA AATI detection peak diagram (Agilent5400) (A) and simulated gel diagram (B) of the DNA product obtained using QIAGEN DNeasy Power Soil Pro kit;

[0041] Figure 4 The DNA AATI detection peak diagram (Agilent5400) (A) and simulated gel diagram (B) of the DNA product obtained by the hand-lift method;

[0042] Figure 5 The DNA AATI detection peak diagram (Agilent5400) (A) and simulated gel diagram (B) of the DNA product obtained using the MP FastDNA Spin Kit for Soil;

[0043] Figure 6 The DNA AATI detection peak diagram (Agilent 5400) (A) and simulated gel diagram (B) of the DNA product obtained from marine sediments at a depth of 180 cm in Example 2 of the present invention;

[0044] Figure 7 The DNA AATI detection peak diagram (Agilent 5400) (A) and simulated gel diagram (B) of the DNA product obtained from marine sediments with a depth of 0-5 cm in Example 2 of the present invention.

[0045] in, Figure 1-Figure 7 In Figure A, the horizontal axis "size (bp)" reflects the fragment distribution of the capillary sample, and the vertical axis "RFU" value refers to the real-time fluorescence signal intensity of the sample during capillary separation. The higher the RFU value, the higher the sample concentration; Low Marker (LM) and Up Marker (UM): refer to the reference objects (not the fragments of the sample itself) analyzed with the sample, which are used to calibrate the fragment size and concentration of the sample. Figure B is a simulated gel image and the corresponding Ladder size value size marking. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0047] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0048] The chemical reagents used in the following examples and their information are as follows:

[0049] (1) Cell lysis buffer:

[0050] 30 mM Tris-HCl, 30 mM disodium EDTA, 1% Triton X-100, 800 mM guanidine hydrochloride, the prepared solution was sterilized by autoclaving twice, and the pH was adjusted to 10.0 with NaOH.

[0051] Place a magnetic stir bar in the solution to ensure that the NaOH crystals and Triton X-100 are completely dissolved. Prepare 250 mL of solution and add 3 g of NaOH. The pH of the solution will be much higher than the target value (pH = 10.0), but after one autoclave, the pH will be significantly lower. If necessary, add an appropriate amount of hydrochloric acid solution (such as 5M) to adjust the pH to about 10.5 and autoclave again. After two autoclaves, the pH is close to 10.0. If adjustment is still needed, fine-tune by adding small amounts of hydrochloric acid or NaOH solution (such as 5M). Gently shake or swirl the solution before use to ensure that the concentration of Triton X-100 is uniform. When adding Triton X-100, cut off the tip of the pipette and aspirate Triton X-100 very slowly, making sure it is completely injected into the cell lysate. If you are unsure how much to add, you can add a little more to ensure that the concentration is at least 0.5%.

[0052] (2) 10 mM sodium hexametaphosphate solution.

[0053] (3) Molecular grade chloroform-isoamyl alcohol mixture (toxic, volatile, prepared in a volume ratio of 24:1).

[0054] (4) Linear polyacrylamide (LPA); store in a refrigerator and avoid exposure to light.

[0055] (5) 5M sodium chloride solution.

[0056] (6) Anhydrous ethanol.

[0057] (7) EZNA Soil DNA Kit from OMEGA.

[0058] Example 1

[0059] A method for extracting DNA from marine sediments comprises the following steps:

[0060] 1. Obtain marine sediments at a depth of 370 cm in the southern sea area, and operate them immediately after collection or freeze them at a temperature below -80°C before extraction.

[0061] 2. Add 0.25 g of marine sediment into a 1.5 mL cryovial and add sterilized zirconium beads with a particle size of 1.8-2.2 mm. The volume of the zirconium beads is 10%-20% of the volume of the cryovial.

[0062] 3. Add 100 μL of 10 mM sodium hexametaphosphate solution to the cryovial containing the marine sediment and zirconium beads, and shake gently to allow the solution to completely wet or coat the marine sediment sample.

[0063] 4. Add another 0.5 mL of cell lysis buffer, invert and mix, and then vortex briefly (10 seconds).

[0064] 5. Place the cryovials in FastPrep-24 TM In the horizontal stand of the 5G rapid sample preparation instrument, set the speed to 7.5m / s and the time to 45s. After the equipment is finished running, place the cryotubes in the Eppendorf Incubate on a FP constant temperature mixer at 50-60°C and 800 rpm for 1 hour.

[0065] 6. After incubation, centrifuge at 10,000 × g, 4°C for 10 min and transfer the supernatant to a clean centrifuge tube, transferring as much supernatant as possible.

[0066] 7. Add an equal volume of cold chloroform-isoamyl alcohol mixture to the supernatant, vortex mix for 10 seconds, and centrifuge at 10,000×g and 4°C for 10 minutes. If the resulting aqueous phase is clear and free of chloroform or particles, proceed directly to the next step; otherwise, repeat this step to ensure that a clear aqueous phase is obtained without chloroform or particles.

[0067] 8. Add LPA to the obtained aqueous phase (containing DNA extract) to a concentration of 20 μg / mL in the aqueous phase. At the same time, add 0.2 times the volume of the aqueous phase 5M sodium chloride solution and mix by inverting upside down 3 times.

[0068] 9. Add 2.5 times the total volume of anhydrous ethanol of the aqueous phase and the sodium chloride solution, mix thoroughly and precipitate at room temperature in the dark for 2 hours.

[0069] 10. After precipitation in the dark, centrifuge at room temperature and 14,000 × g for 20 min, discard the supernatant, and there will be a small amount of precipitation on the bottom wall of the centrifuge tube.

[0070] 11. The following steps require the use of OMEGA's EZNA Soil DNA Kit.

[0071] 12. Add 200 μL Elution Buffer to the centrifuge tube containing the precipitate, so that it can immerse the sediment on the bottom wall of the centrifuge tube, vortex gently for 10 seconds, and place in Eppendorf Incubate in FP constant temperature mixer at 70°C and 600 rpm for 10-20 min to dissolve the precipitate.

[0072] 13. Add 100 μL cHTR Reagent to the incubated centrifuge tube and vortex to mix (10 seconds). Before using cHTRReagent, shake the bottle thoroughly to fully resuspend the cHTR Reagent.

[0073] 14. Place at room temperature for 2 minutes, centrifuge at maximum speed for 2 minutes, and transfer the supernatant to a new 2 mL centrifuge tube. If the supernatant is still dark in color, repeat steps 13-14 for a second cHTRReagent treatment.

[0074] 15. Add an equal volume of XP1 Buffer to the supernatant and vortex mix (10 seconds) to obtain a mixed solution.

[0075] 16. Put Place the DNA Mini Column in a new 2 mL collection tube and transfer the mixture from step 15 to Centrifuge the DNA Mini Column at 10,000 × g for 1 min at room temperature and discard the filtrate.

[0076] 17. After step 16 Place the DNA Mini Column in a new 2 mL collection tube and add 700 μL HBC Buffer to Centrifuge the DNA Mini Column at 10,000 × g for 1 min at room temperature and discard the filtrate.

[0077] It should be noted that before use, please add isopropanol to dilute HBC Buffer according to the instructions.

[0078] 18. After step 17 Place the DNA Mini Column in a new 2 mL collection tube and add 700 μL DNA Wash Buffer to Centrifuge the DNA Mini Column at 10,000 × g for 1 min at room temperature and discard the filtrate.

[0079] It should be noted that DNA Wash Buffer must be diluted with anhydrous ethanol before use.

[0080] 19. After step 18 DNA Mini Column, room temperature, empty column 13000 × g centrifugation for 2 minutes, dry the column. This step is very critical to remove impurities that inhibit downstream experiments.

[0081] 20. After step 19 Transfer the DNA Mini Column to a clean 1L centrifuge tube, add 0-100 μL Elution Buffer preheated to 70°C to the center of the column membrane, leave at room temperature for 1-2 minutes, centrifuge at 13000×g for 1 minute, and collect the filtrate.

[0082] If a higher concentration of DNA is required, the amount of Elution Buffer can be appropriately reduced and the column can be incubated at 65°C, which can improve the DNA elution efficiency compared to incubation at room temperature.

[0083] 21. Repeat step 20 for a second elution and collect the filtrate.

[0084] 22. Discard the column and store the filtrate obtained in step 20 and step 21, i.e. the DNA product, at -20°C.

[0085] Five different methods and kits were used to extract microbial genomic DNA from deep ocean sediment samples (370 cm) from the same source. The five methods were the method of the present invention (i.e., Example 1), OMEGA EZNA Soil DNA Kit, QIAGEN DNeasy Power Soil Pro kit, a portable method, and MP FastDNA Spin Kit for Soil. The extracted DNA samples were then sent to Beijing Novogene Technology Co., Ltd. for electrophoresis. The results were as follows: Figure 1-Figure 5 And as shown in Table 1.

[0086] It should be noted that when using OMEGA EZNA Soil DNA Kit, QIAGEN DNeasy Power SoilPro kit and MP FastDNA Spin Kit for Soil to extract DNA from marine sediments, just follow the steps described in the instructions of each reagent.

[0087] The steps of the hand-carrying method are steps 1 to 10 in Example 1.

[0088] Table 1 Results of DNA extraction from marine sediments using Example 1 and other methods

[0089]

[0090]

[0091] As can be seen from Table 1, by extracting DNA from marine sediments using the method of the present invention, more DNA with higher concentration can be obtained, which can better meet the requirements of metagenomic sequencing and PCR reaction.

[0092] Example 2

[0093] The method of Example 1 was used to extract DNA from sediments at different depths at the same location. The results are shown in Table 2. Figure 6 , 7 The DNA AATI detection peak graph (Agilent5400) fed back by the sequencing company.

[0094] Table 2 DNA extraction results of sediments at different depths at the same location

[0095]

[0096] In this example, DNA was extracted from sediments at the middle layer (180 cm) and the surface layer (0-5 cm) at the same location, with concentrations of 3.87 ng / μL and 4.02 ng / μL, respectively, and total amounts of 0.23214 μg and 0.22501 μg, respectively.

[0097] The method provided by the present invention is universal and suitable for extracting DNA from marine sediments of different depths. It can successfully extract more DNA with higher concentration from sediments more than 3 meters deep, which can better meet the requirements of metagenomic sequencing and PCR reaction.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting DNA from marine sediments, characterized in that: include: Sodium hexametaphosphate and cell lysate are added to the marine sediment, mixed, incubated, and centrifuged to obtain a supernatant; Add chloroform-isoamyl alcohol mixture to the supernatant, mix well, centrifuge, remove the organic phase, and obtain the aqueous phase; Linear polyacrylamide and sodium chloride were added to the aqueous phase, and ethanol was added after mixing. The mixture was precipitated in the dark and centrifuged to obtain a DNA extract.

2. The method according to claim 1, characterized in that The method further comprises adding grinding balls to the marine sediment; Preferably, the grinding balls are zirconium beads, the particle size of the zirconium beads is 1.8-2.2 mm, and the volume of the zirconium beads is 10%-20% of the volume of the device used.

3. The method according to claim 1, characterized in that The dosage ratio of the sodium hexametaphosphate, cell lysate and marine sediment is 0.8-1.2nmol:0.4-0.6mL:0.2-0.3g; The sodium hexametaphosphate is prepared into a solution of 8-12 mM and then added into the marine sediment; The cell lysis solution comprises: 28-32 mM Tris-HCl, 28-32 mM disodium EDTA, 1% Triton X-100, 790-810 mM guanidine hydrochloride, and a pH of 9.8-10.

2.

4. The method according to claim 3, characterized in that The cell lysis solution consists of: 30 mM Tris-HCl, 30 mM disodium EDTA, 1% Triton X-100, 800 mM guanidine hydrochloride, and a pH of 10.

0.

5. The method according to claim 1, characterized in that Add an equal volume of chloroform-isoamyl alcohol mixture to the supernatant; Preferably, an equal volume of a chloroform-isoamyl alcohol mixture is added to the supernatant, mixed and centrifuged, and if the aqueous phase is clear and free of chloroform or particles, the obtained aqueous phase is subjected to the next step of treatment; otherwise, the steps of adding an equal volume of a chloroform-isoamyl alcohol mixture to the obtained aqueous phase and mixing and centrifuging are repeated until a clear aqueous phase free of chloroform or particles is obtained.

6. The method according to claim 1 or 5, characterized in that In the chloroform-isoamyl alcohol mixed solution, the volume ratio of chloroform to isoamyl alcohol is 22-26:1, preferably 24:

1.

7. The method according to claim 1, characterized in that The concentration of the linear polyacrylamide in the aqueous phase is 18-22 μg / mL, preferably 20 μg / mL.

8. The method according to claim 1, characterized in that The sodium chloride is added to the aqueous phase in the form of a 4.8-5.2M sodium chloride solution, and the amount of the sodium chloride solution added is 0.15-0.25 times the volume of the aqueous phase; The amount of ethanol added is 2-3 times, preferably 2.5 times, of the total volume of the aqueous phase and the sodium chloride solution; preferably, the ethanol is anhydrous ethanol.

9. The method according to claim 1, characterized in that The method further comprises the step of purifying the DNA extract; Wherein, the purification includes purifying the obtained DNA extract using a purification kit; Preferably, the purified DNA extract is stored at -20°C to -80°C.

10. The method according to claim 9, characterized in that The purification kit includes EZNA Soil DNA Kit.

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