A method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation

Abstract

This invention belongs to the field of biotechnology, specifically relating to a method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation. DNA samples are subjected to ultra-high-speed density gradient centrifugation to separate layers. Sodium acetate, a nucleic acid precipitation aid, and ethanol are added to each layer. Each layer mixture is then filtered through a nucleic acid purification column to obtain DNA. The filtrate from the nucleic acid purification column is subjected to precipitation treatment to recover cesium chloride. This invention simplifies the purification process and shortens the overall operation time.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation. Background Technology

[0002] DNA-SIP is a technique that uses stable isotope labeling to reveal functional microorganisms involved in the metabolic processes of labeled substrates in complex environmental microbial communities. It uses stable isotopes such as 13C to label substrates in a culture environment. The environmental microbial cells that use the labeled substrates continuously divide, grow, reproduce, and synthesize 13C-DNA. The total genomic DNA of the environmental microorganisms is extracted, and the 13C-DNA is separated from the 12C-DNA by ultra-high-speed density gradient centrifugation. Further analysis of the 13C-DNA using molecular biology techniques can reveal the functional microorganisms in complex environmental samples that have assimilated the labeled substrates.

[0003] In existing DNA-SIP processes, after DNA is centrifuged and separated into layers at ultrahigh speed, the most common DNA purification method is precipitation with polyethylene glycol and anhydrous ethanol. This method is cumbersome, time-consuming, and labor-intensive, resulting in low DNA recovery rates, significant DNA loss, poor reproducibility among different experimenters, and the potential for errors that could lead to DNA failure. Furthermore, cesium chloride is expensive, and traditional polyethylene glycol and anhydrous ethanol precipitation methods cannot recover it, resulting in the waste of cesium chloride after the experiment. Therefore, providing a DNA purification method that overcomes the limitations of the above methods would be more reliable, faster, and more convenient for DNA-SIP experiments, while simultaneously recovering cesium chloride for reuse, greatly reducing experimental costs. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation, thereby solving the problem of low DNA purification and recovery rates in current DNA-SIP experiments.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for DNA purification and cesium chloride recovery based on ultra-high speed density gradient centrifugation is disclosed. The DNA sample is subjected to ultra-high speed density gradient centrifugation to separate layers. Sodium acetate, nucleic acid precipitation aid and ethanol are added to each layer respectively. The mixture of each layer is filtered through a nucleic acid purification column to obtain DNA. The filtrate of the nucleic acid purification column is subjected to precipitation treatment to recover cesium chloride.

[0006] Conditions for ultra-high speed density gradient centrifugation: rotation speed 190000g, time 44h.

[0007] Sodium acetate was added at 2.5%-3% of the volume of each DNA sample layer, nucleic acid precipitation aid was added at 0.25%-0.3% of the volume of each DNA sample layer, and ethanol was added at 1.4-1.45 times the volume of each DNA sample layer.

[0008] Nucleic acid purification column filtration is performed as follows: each layer of the mixture is transferred to a nucleic acid purification column, the filtrate is collected, ethanol is added to the nucleic acid purification column for washing, TE buffer is added to the washed nucleic acid purification column, and the DNA is eluted by centrifugation after standing at 20℃-25℃.

[0009] Collect the filtrate filtered from the nucleic acid purification column, add anhydrous ethanol to the filtrate, centrifuge and discard the supernatant, take the cesium chloride precipitate, and dry the cesium chloride precipitate to obtain cesium chloride solid.

[0010] Specifically, the steps include the following: (1) The DNA sample was separated into layers by ultra-high speed density gradient centrifugation, and sodium acetate, nucleic acid precipitation aid and ethanol were added to each layer to obtain a mixture; (2) Place the nucleic acid purification column on the purification device connected to the vacuum pump; (3) Transfer the mixture of each layer obtained in step (1) to a nucleic acid purification column, turn on the vacuum pump to filter, and collect the filtrate; (4) Add ethanol to the nucleic acid purification column of step (3) for washing, and turn on the vacuum pump until the ethanol is completely filtered; repeat this washing step at least once. (5) Centrifuge the nucleic acid purification columns cleaned in step (4) respectively; (6) Add TE buffer to the center of the nucleic acid purification column membrane in step (5), let stand, centrifuge, and elute to recover DNA; (7) Collect the filtrate produced in step (3), add anhydrous ethanol to the filtrate, centrifuge, discard the supernatant, take the cesium chloride precipitate, dry the cesium chloride precipitate to obtain cesium chloride solid; dissolve the cesium chloride solid in sterile enzyme-free water and use it again for ultra-high speed density gradient centrifugation.

[0011] Step (4) Centrifugation conditions: 14000g-15000g for 2min.

[0012] Step (5) Centrifugation conditions: 14000g-15000g for 2min.

[0013] Step (6): Add TE buffer to the center of the nucleic acid purification column membrane from step (5), let stand at 20℃-25℃ for 10 minutes, centrifuge at 14000g-15000g for 1 minute, and elute and recover the DNA.

[0014] Step (7) Centrifugation conditions: 8000rpm-10000rpm for 10min; drying conditions: 40℃-50℃ for 60h-72h.

[0015] The advantages of this invention are: This invention involves adding sodium acetate, a nucleic acid precipitation aid, and ethanol to a DNA sample. Sodium acetate helps adjust the sample pH to allow the nucleic acid purification column to better adsorb DNA, the nucleic acid precipitation aid can effectively precipitate and separate DNA from the cesium chloride solution, and ethanol can clean and filter impurities from the nucleic acid purification column.

[0016] This invention uses a nucleic acid purification column and purification device for filtration to separate and remove cesium chloride-containing filtrate. DNA is retained in the nucleic acid purification column and eluted for recovery. Anhydrous ethanol is added to the cesium chloride solution to generate cesium chloride precipitate. After centrifugation, drying, and recrystallization, high-purity cesium chloride solid is obtained. The high-purity cesium chloride solid can be reused for ultra-high-speed density gradient centrifugation, which significantly reduces the cost of consumables.

[0017] This invention simplifies the operation steps of traditional purification processes, shortens the overall operation time, and thus improves the efficiency of experimental work. It does not require expensive equipment and reagents, and can significantly improve the recovery rate and purity of DNA, demonstrating good practicality and application prospects. Attached Figure Description

[0018] Figure 1 This is an electrophoretic detection image of purified DNA from Example 1 of the present invention. Detailed Implementation

[0019] The technical solution of the present invention is further described below with reference to specific implementation examples, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0020] Unless otherwise specified, the instruments, raw materials and reagents used in this invention can all be obtained commercially, including nucleic acid precipitation aid (Cooler SL3210), nucleic acid purification column (Xinjing 7204050), and purification device (Xinjing A-100).

[0021] The DNA samples were obtained from soil samples taken from a cornfield.

[0022] DNA samples were extracted using the MP SPINeasy DNA Pro Kit for Soil extraction kit.

[0023] Example 1 (1) Centrifuge the tubes containing the DNA sample at 190,000 g for 44 hours using a Beckman Optima centrifuge. TMAfter dividing the L-100 XP centrifuge tube (rotor model Vti65.2, centrifuge tube model Beckman Ultra-Clear 344075) into 15 equal layers with a volume of 350µL per layer, each layer was further divided into 10µL sodium acetate (3M, pH 5.2, sterile and enzyme-free), 1µL nucleic acid precipitation aid, and 500µL ethanol (70%). (2) The nucleic acid purification column is placed on the purification device, which is connected to a vacuum pump; (3) After shaking the 15-layer mixture obtained in step (1) by hand, transfer it to 15 nucleic acid purification columns, turn on the vacuum pump, filter and collect the filtrate containing cesium chloride in the purification device; (4) Add 700µL of ethanol (70%) to the nucleic acid purification column of step (3), turn on the vacuum pump, and after all the ethanol in the column has been filtered, repeat the washing process once more. (5) Transfer the cleaned nucleic acid purification column from step (4) to a clean 2mL centrifuge tube and centrifuge at 15000rpm for 2min. (6) Discard the 2mL centrifuge tube and replace it with a sterile, enzyme-free 1.5mL centrifuge tube. Add 50µL LTE buffer to the center of the membrane of the purification column, incubate at 20℃-25℃ for 10 minutes, centrifuge at 12000rpm for 1 minute to elute and recover the DNA. See the DNA electrophoresis detection image below. Figure 1 DNA purity is shown in Table 1; (7) Collect the filtrate produced by each nucleic acid purification column in step (3), add 5 times the volume of anhydrous ethanol, centrifuge at 8000 rpm for 10 min, discard the supernatant, recover the cesium chloride precipitate at the bottom of the centrifuge tube, transfer the cesium chloride precipitate to a 50℃ oven and dry for 72 h to obtain cesium chloride solid.

[0024] Cesium chloride solid was dissolved in sterile, enzyme-free water and then used again for ultra-high-speed density gradient centrifugation.

[0025] Comparative Example 1 Take 15 layers of the same sample as in Example 1, add 550µL of polyethylene glycol 6000 solution to each layer, invert the sample several times to mix the solution, let it stand at 25°C for 2 hours to precipitate DNA, centrifuge at 15000rpm at 20°C for 30 minutes, and remove the supernatant.

[0026] Add 500µL of ethanol (70%) to wash the DNA precipitate, centrifuge at 15000rpm for 10min, remove the supernatant, repeat this step (to further remove chloroethylene and polyethylene glycol 6000), precipitate the DNA at 25°C, and after ensuring that there is no liquid in the DNA precipitate, dissolve it in 50µL LTE buffer and store it.

[0027] The DNA obtained from the above examples and comparative examples was tested, and the test results are shown in Table 1.

[0028] Table 1

[0029] DNA purity can be evaluated using OD260 / 280 and OD260 / 230 as indicators. Pure DNA has an OD260 / 280 of 1.8-2.0 and an OD260 / 230 greater than 2.0. After ultra-high-speed density gradient centrifugation, DNA concentrates in layers 8, 9, and 10; therefore, only data from layers 8, 9, and 10 are used in the experimental results. As shown in Table 1, compared to Comparative Example 1, the purification method in Example 1 not only has the highest DNA recovery rate but also the optimal OD260 / 280. The purification method in Comparative Example 1 yields a lower total amount of DNA with poorer purity. Therefore, this invention adds sodium acetate, a nucleic acid precipitation aid, and ethanol to the DNA sample. Sodium acetate helps adjust the sample pH, allowing the nucleic acid purification column to better adsorb DNA, while the nucleic acid precipitation aid effectively precipitates and separates DNA from the cesium chloride solution.

Claims

1. A method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation, characterized in that, DNA samples were separated into layers by ultra-high-speed density gradient centrifugation. Sodium acetate, nucleic acid precipitation aid, and ethanol were added to each layer. Each layer mixture was then filtered through a nucleic acid purification column to obtain DNA. The filtrate from the nucleic acid purification column was subjected to precipitation treatment to recover cesium chloride.

2. The method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation according to claim 1, characterized in that, Sodium acetate was added at 2.5%-3% of the volume of each DNA sample layer, nucleic acid precipitation aid was added at 0.25%-0.3% of the volume of each DNA sample layer, and ethanol was added at 1.4-1.45 times the volume of each DNA sample layer.

3. The method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation according to claim 1, characterized in that, Nucleic acid purification column filtration is performed as follows: each layer of the mixture is transferred to a nucleic acid purification column, the filtrate is collected, ethanol is added to the nucleic acid purification column for washing, TE buffer is added to the washed nucleic acid purification column, and the DNA is eluted by centrifugation after standing at 20℃-25℃.

4. The method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation according to claim 1, characterized in that, Collect the filtrate filtered from the nucleic acid purification column, add anhydrous ethanol to the filtrate, centrifuge and discard the supernatant, take the cesium chloride precipitate, and dry the cesium chloride precipitate to obtain cesium chloride solid.

5. The method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation according to claim 1, characterized in that, Includes the following steps: (1) The DNA sample was separated into layers by ultra-high speed density gradient centrifugation, and sodium acetate, nucleic acid precipitation aid and ethanol were added to each layer to obtain a mixture; (2) Place the nucleic acid purification column on the purification device connected to the vacuum pump; (3) Transfer the mixture of each layer obtained in step (1) to a nucleic acid purification column, turn on the vacuum pump to filter, and collect the filtrate; (4) Add ethanol to the nucleic acid purification column of step (3) for washing, and turn on the vacuum pump until the ethanol is completely filtered; repeat this washing step at least once. (5) Centrifuge the nucleic acid purification columns cleaned in step (4) respectively; (6) Add TE buffer to the center of the nucleic acid purification column membrane in step (5), let stand, centrifuge, and elute to recover DNA; (7) Collect the filtrate produced in step (3), add anhydrous ethanol to the filtrate, centrifuge, discard the supernatant, take the cesium chloride precipitate, dry the cesium chloride precipitate to obtain cesium chloride solid; dissolve the cesium chloride solid in sterile enzyme-free water and use it again for ultra-high speed density gradient centrifugation.

6. The method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation according to claim 5, characterized in that, The centrifugation conditions for step (4) are: centrifuge at 14000g-15000g for 2min; the centrifugation conditions for step (5) are: centrifuge at 14000g-15000g for 2min.

7. The method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation according to claim 5, characterized in that, Step (6): Add TE buffer to the center of the nucleic acid purification column membrane from step (5), let stand at 20℃-25℃ for 10 minutes, centrifuge at 14000g-15000g for 1 minute, and elute and recover the DNA.

8. The method for DNA purification and cesium chloride recovery based on ultra-high-speed density gradient centrifugation according to claim 5, characterized in that, Step (7) Centrifugation conditions: 8000rpm-10000rpm for 10min; drying conditions: 40℃-50℃ for 60h-72h.

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