Method for simultaneously extracting DNA and RNA

Abstract

The invention discloses a method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and relates to the technical field of molecular biology, the method comprises the following steps: sample lysis incubation: co-incubating a biological sample with a lysis buffer solution, a reducing agent and proteolytic enzyme; adding a binding solution, namely adding the binding solution into the mixture subjected to lysis incubation, wherein the binding solution at least comprises a chaotropic agent, a nonionic surfactant and alcohol; according to the method, the biological sample is incubated together with the cracking buffer solution which does not contain chaotropic agent and chelating or complexing agent, the reducing agent and the proteolytic enzyme, and the anionic surfactant and the buffer compound in the cracking buffer solution have a synergistic effect and are matched with the reducing agent and the proteolytic enzyme, so that the biological sample can be efficiently cracked without physical crushing treatment; and even complex samples such as fiber tissues and high cell content can be fully cracked, so that DNA and RNA released from the samples are more complete, and nucleic acid loss and breakage caused by insufficient cracking are reduced.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically a method for simultaneously extracting DNA and RNA. Background Technology

[0002] In the field of molecular pathology, such as in the diagnosis or treatment of tumors, DNA and RNA analysis are commonly used methods. However, the sample size obtained from clinical practice is usually too small to extract DNA or RNA independently. Furthermore, since some samples are not homogeneous, splitting samples often cannot guarantee that tumor cells or target nucleic acids are split in the same proportion. Therefore, it is very important to extract DNA and RNA simultaneously from a single, unseparated sample.

[0003] Extracting DNA and RNA using organic solvents is a common method in this field. However, since phenol is a toxic reagent, a safer and more effective solid-phase capture method has been invented. The principle of this method is that nucleic acids can be adsorbed by solid materials, such as silica, in a certain solution environment. In a solution containing a dissociating agent and / or alcohol, nucleic acids come into contact with and bind to the solid material, while impurities such as proteins and lipids remain in the solution. Subsequently, the solid material is washed once or multiple times to remove impurities other than nucleic acids. Finally, with the help of a suitable solution environment, the nucleic acids are eluted from the solid material. The general operation for simultaneously extracting DNA and RNA using the solid-phase capture method is as follows: First, the biological sample is physically broken up. The lysed sample is incubated in a lysis buffer containing a dissociating agent and a reducing agent. Then, the lysate is brought into contact with the first solid phase. DNA binds to the first solid phase, while RNA remains in the solution. Then, alcohol is added to the remaining solution, and the resulting mixture is brought into contact with the second solid phase. RNA binds to the second solid phase. After washing the two solid phases separately, DNA is eluted from the first solid phase and RNA is eluted from the second solid phase.

[0004] While the above methods avoid the use of toxic reagents and sample splitting, they still have several drawbacks. First, the physical disruption of biological samples is cumbersome. Second, relying on two separate solid phases for capture and purification is not only time-consuming and costly, but also results in the dilution of nucleic acids due to the separate elution of DNA and RNA. Finally, the lysis buffer contains a high concentration of ionizing agent to inactivate RNase, which severely inhibits the activity of proteolytic enzymes. In the absence of proteolytic enzyme digestion, it is difficult to lyse samples with high fibrous, cellular, or protein content. In incompletely lysed samples, protein and cell debris cross-link with nucleic acids and adsorb onto the solid phase surface, leading to low purity of extracted nucleic acids and inhibiting downstream detection. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for simultaneously extracting DNA and RNA. This method enables the simultaneous extraction of DNA and RNA using a single solid phase, greatly simplifying the operation process and reducing experimental costs. Regarding sample lysis, by co-incubating biological samples with a specific lysis buffer, reducing agent, and proteolytic enzyme, the lysis buffer, which does not contain dissociative agents or chelating or complexing agents but rather anionic surfactants and buffer compounds, combined with the action of the reducing agent and proteolytic enzyme, eliminates the need for physical fragmentation of the sample. This allows for the effective extraction of DNA and RNA from complex samples such as fibrous tissue and high-cell-content samples. Furthermore, the nucleic acids extracted by this method have higher yield and purity, less inhibition of downstream applications, and significantly improved detection sensitivity.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for simultaneously extracting DNA and RNA, the method comprising the following specific steps:

[0007] Sample lysis incubation: Incubate biological samples with lysis buffer, reducing agent and proteolytic enzymes;

[0008] Add binding solution: Add binding solution to the mixture after pyrolysis incubation. The binding solution contains at least one liquid release agent, one nonionic surfactant and one alcohol.

[0009] Nucleic acid binding solid phase: Utilizing the adsorption properties of silicon-containing solid phase materials for nucleic acids in a solution environment of pH 6-9, DNA and RNA are bound to a single solid phase;

[0010] Washing and elution: After washing the solid phase with washing solution to remove impurities, DNA and RNA are eluted from the solid phase using elution solution.

[0011] Furthermore, in the sample lysis incubation step, the lysis buffer contains at least one buffer compound and at least one anionic surfactant.

[0012] Furthermore, the buffer compound is preferably selected from N-(tris(hydroxymethyl)methyl)glycine TRICINE, tris(hydroxymethyl)aminomethane TRIS, di(2-hydroxyethyl)iminotris(hydroxymethyl)methane BIS-TRIS, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) HEPES, N-cyclohexyl-2-aminoethanesulfonic acid CHES, N,N-bis(2-hydroxyethyl)glycine BICINE, 2-(N-morpholino)ethanesulfonic acid MES, piperazine-1,4-bis(2-ethanesulfonic acid) PIPES, 3-(N-morpholino)propanesulfonic acid MOPS, phosphate buffers, and the buffer compound is present at a concentration ≤500 mM.

[0013] Furthermore, the anionic surfactant is preferably a bile acid-based detergent, a sarcosine-based detergent, a sulfate or sulfonate of a fatty alcohol, and the anionic surfactant is present at 0.1% to 20% w / v.

[0014] Furthermore, the reducing agent is preferably selected from dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, dithioerythritol, sodium thiosulfate, β-mercaptoethanol, or a mixture thereof.

[0015] Furthermore, the proteolytic enzyme is a protease, optionally a serine protease, present at a concentration ranging from 0.001 to 5% w / v, and the incubation temperature is set to 25–70°C, preferably 30–60°C.

[0016] Furthermore, in the step of adding the binding liquid, the liquid release agent is preferably a sodium salt or guanidine salt liquid release agent, that is, a mixture of two or more of sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, and guanidine isothiocyanate.

[0017] Furthermore, in the step of adding the binding liquid, the nonionic surfactant is preferably a polyoxyethylene-based nonionic detergent, including polyoxyethylene fatty acid esters and polyoxyethylene alkyl phenyl ethers, and the nonionic surfactant is present at a concentration in the range of 0.1% to 10% w / v.

[0018] Furthermore, in the step of adding the binding liquid, the alcohol is preferably a branched or unbranched alcohol having 1-11 carbon atoms, most preferably selected from ethanol, ethylene glycol, isopropanol, n-butanol, and 1,3-butanediol, present at a concentration in the range of 1% to 75% w / v.

[0019] Furthermore, in the washing and elution steps, the solid phase is washed 1 to 3 times with 80% ethanol to remove residual elution agent, protein and salt ions. During elution, an RNase-free water elution buffer is used to destroy the binding force between nucleic acids and the solid phase in a low-salt environment, thereby eluting DNA and RNA.

[0020] Compared with existing technologies, this method for simultaneously extracting DNA and RNA has the following advantages:

[0021] I. This invention involves co-incubating biological samples with a lysis buffer, a reducing agent, and proteolytic enzymes that do not contain cleavage agents or chelating or complexing agents. The anionic surfactants and buffer compounds in the lysis buffer work synergistically with the reducing agent and proteolytic enzymes to efficiently lyse biological samples without physical fragmentation. Even complex samples such as fibrous tissue and high cell content can be fully lysed, resulting in more intact DNA and RNA released from the sample and reducing nucleic acid loss and breakage caused by incomplete lysis.

[0022] Second, this invention extracts DNA and RNA simultaneously without the need to segment a single biological sample, avoiding operational errors that may result from sample segmentation and the difficulty in ensuring proportional segmentation of tumor cells or target nucleic acids in the sample. At the same time, the entire extraction process only requires the use of a single solid material, which greatly simplifies the experimental steps, significantly saves experimental time, reduces labor costs, and improves experimental efficiency.

[0023] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0025] Figure 1 This is a schematic diagram of agarose gel electrophoresis;

[0026] Figure 2 This is a flowchart of a method for simultaneously extracting DNA and RNA. Detailed Implementation

[0027] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0028] Example 1

[0029] Cell sample: Cultured human HeLa cells;

[0030] Lysis buffer A formulation: 10% (w / v) N-lauroyl sarcosinate sodium, 2.5% (w / v) tris(hydroxymethyl)aminomethane (TRIS), adjust pH to 8.0 with hydrochloric acid, and add purified water to make up to volume;

[0031] Other reagents: PBS buffer, RNase-free water.

[0032] Experimental steps:

[0033] Cell processing: HeLa cells in logarithmic growth phase were washed twice with PBS, centrifuged at 500g for 3 minutes to collect the cells, and the cells were divided into two equal parts.

[0034] Sample preservation: Add 200 μL of lysis buffer A to the first batch of cells, gently pipette to mix, and store in a 37°C incubator; do not add any reagents to the second batch of cells and store directly in a -20°C freezer.

[0035] Nucleic acid extraction: Three days later, nucleic acid was extracted from the two samples. For the first sample, the lysis buffer was directly used for subsequent operations. For the second sample, the cells were resuspended in 200 μL PBS and then lysed.

[0036] Nucleic acid quantification: The extracted DNA and RNA were quantitatively detected using a Qubit4 fluorometer, with each sample tested three times.

[0037] Experimental results:

[0038]

[0039]

[0040] Results analysis: Compared with cell samples stored at -20℃, the DNA and RNA content in cell samples stored at 37℃ with lysis buffer A did not decrease significantly, and the detection results of each replicate sample showed little difference. This indicates that lysis buffer A can effectively inhibit the activity of DNase and RNase at higher temperatures, prevent nucleic acid degradation, and provide a stable sample environment for subsequent nucleic acid extraction.

[0041] Example 2

[0042] Materials and reagents:

[0043] Tissue samples: Mouse muscle tissue, cut into three weight specifications of 0.2mg, 2mg and 20mg, with 3 portions of each specification prepared;

[0044] Lysis buffer A formulation: 10% (w / v) N-lauroyl sarcosinate sodium, 2.5% (w / v) TRIS, pH 8.0;

[0045] Lysis buffer B formulation: 55% (w / v) guanidine isothiocyanate, 2.5% (w / v) TRIS, pH 8.0;

[0046] The binding buffer formulation is: 40% (w / v) guanidine hydrochloride, 50% (w / v) isopropanol, and 5% (w / v) Triton X-100.

[0047] Other reagents: β-mercaptoethanol, proteinase K, PBS buffer.

[0048] Experimental steps:

[0049] Sample preparation: Place mouse muscle tissue into 1.5 mL centrifuge tubes and label them as Group 1 (using lysis buffer A) and Group 2 (using lysis buffer B);

[0050] Lysis treatment: Add 200 μL of lysis buffer A to each tube of the first group and 200 μL of lysis buffer B to each tube of the second group. Then add 10 μL of β-mercaptoethanol and 20 μL of proteinase K to each tube of both groups and vortex to mix.

[0051] Incubation process: Place the centrifuge tubes in a 37°C constant temperature metal bath and incubate for 1 hour, vortexing once every 15 minutes to ensure that the sample is fully lysed;

[0052] Binding treatment: After incubation, add 400 μL of binding buffer to each tube of each group, vortex for 1 minute to ensure the solution is fully mixed;

[0053] Nucleic acid adsorption: The obtained solution was transferred to a nucleic acid adsorption column (the adsorption column filter membrane is composed of three layers of GF / B glass fiber with a pore size of 1.0 μm), centrifuged at 10000×g for 1 minute, and the passage of the solution through the filter membrane was observed and recorded.

[0054] Experimental results:

[0055]

[0056] Results Analysis: In experiments with 2 mg and 20 mg mouse muscle tissue, samples treated with lysis buffer A completely passed through the nucleic acid adsorption column filter membrane, indicating thorough lysis without residual tissue debris clogging the membrane. Samples treated with lysis buffer B, however, showed incomplete passage, suggesting that the high concentration of guanidine isothiocyanate inhibited proteinase K activity, leading to incomplete lysis. Residual protein and cell debris then cross-linked with nucleic acids, clogging the membrane. For small samples (0.2 mg), both lysis buffers allowed complete passage. This is because the smaller sample volume meant that even with incomplete lysis, the membrane was less likely to clog. These results demonstrate that lysis buffer A effectively improves the lysis capacity for samples with high fiber and cell content, overcoming the shortcomings of traditional lysis buffers.

[0057] Example 3

[0058] Materials and reagents:

[0059] Tissue samples: Mouse muscle tissue, homogenized and divided into 6 equal parts, numbered 1-6;

[0060] Lysis buffer A formulation: 10% (w / v) N-lauroyl sarcosinate sodium, 2.5% (w / v) TRIS, pH 8.0;

[0061] Lysis buffer B formulation: 55% (w / v) guanidine isothiocyanate, 2.5% (w / v) TRIS, pH 8.0;

[0062] The binding buffer formulation is: 40% (w / v) guanidine hydrochloride, 50% (w / v) isopropanol, and 5% (w / v) Triton X-100.

[0063] Other reagents: β-mercaptoethanol, proteinase K, 80% ethanol, RNase-free water, DNA adsorption column, RNA adsorption column.

[0064] Experimental steps:

[0065] Samples 1-3:

[0066] Add 200 μL of lysis buffer A, 10 μL of β-mercaptoethanol, and 20 μL of proteinase K to the sample and incubate at 37°C for 15 minutes.

[0067] Add 450 μL of binding buffer to the solution and vortex to mix.

[0068] Transfer the solution to a DNA / RNA adsorption column and centrifuge at 10000×g for 1 minute to allow the solution to pass completely through the filter membrane.

[0069] The adsorption column was washed three times with 80% ethanol, and centrifuged at 10,000 × g for 1 minute each time.

[0070] DNA and RNA were eluted from the adsorption column using 50 μL of RNase-free water, and the eluent was collected.

[0071] Samples 4-6:

[0072] Add 200 μL of lysis buffer B, 10 μL of β-mercaptoethanol, and 20 μL of proteinase K to the sample and incubate at 37°C for 15 minutes.

[0073] Transfer the solution to a DNA adsorption column, centrifuge at 10000×g for 1 minute, and collect the filtrate.

[0074] Add 450 μL of ethanol to the filtrate and vortex to mix.

[0075] Transfer the solution to an RNA adsorption column and centrifuge at 10000×g for 1 minute to allow the solution to pass completely through the filter membrane.

[0076] Wash the DNA adsorption column and RNA adsorption column three times with 80% ethanol, centrifuging at 10000×g for 1 minute each time.

[0077] DNA and RNA were eluted from the DNA adsorption column and RNA adsorption column respectively using 50 μL of RNase-free water, and the eluent was collected.

[0078] Detection method:

[0079] Micro-volume UV spectrophotometric detection: The concentration (ng / μL) of nucleic acid in the eluent and the ratios of 260 / 280 and 260 / 230 were detected using a micro-volume UV spectrophotometer to assess the purity of the nucleic acid;

[0080] Qubit Quantitative Detection: Precise quantification of DNA and RNA using the Qubit4 fluorometer;

[0081] Agarose gel electrophoresis detection: Prepare a 1% agarose gel, take 10 μL of nucleic acid sample for electrophoresis, and observe the integrity and clarity of the nucleic acid bands under ultraviolet light. Figure 1 As shown.

[0082] Experimental results:

[0083]

[0084] Results Analysis: Regarding nucleic acid concentration, the DNA concentration extracted by the method of this invention was between 31.8-41.8 ng / μL, and the RNA concentration was between 68.0-69.8 ng / μL, while the DNA concentration extracted by the traditional two-phase method was only 3.4-5.2 ng / μL, and the RNA concentration was 10.0-12.3 ng / μL. The nucleic acid yield of the method of this invention is significantly higher than that of the traditional method. In terms of purity, the 260 / 280 ratio of the nucleic acid extracted by the method of this invention is close to 2.0, and the 260 / 230 ratio is greater than 2.0, indicating high nucleic acid purity. The nucleic acid extracted by the present invention has a high yield and is almost free of protein and salt ion contamination. In contrast, the nucleic acid extracted by traditional methods has a 260 / 280 ratio lower than 2.0 and a 260 / 230 ratio even lower, indicating a greater amount of impurity contamination. Agarose gel electrophoresis results show that the DNA and RNA bands extracted by the present invention are clear and complete, with no obvious degradation. In contrast, the nucleic acid bands extracted by traditional methods are blurry and diffuse, indicating a certain degree of degradation. In summary, the present invention not only increases the yield of nucleic acids but also significantly improves their purity and integrity, making it more suitable for downstream molecular biology detection.

[0085] Example 4

[0086] Materials and reagents:

[0087] Sample source: 22 BRAF gene mutant, 3 BRAF gene wild type, and 3 CCDC6-RET gene fusion type thyroid nodule biopsy samples;

[0088] Lysis buffer A formulation: 10% (w / v) N-lauroyl sarcosinate sodium, 2.5% (w / v) TRIS, pH 8.0;

[0089] The binding buffer formulation is: 40% (w / v) guanidine hydrochloride, 50% (w / v) isopropanol, and 5% (w / v) Triton X-100.

[0090] Other reagents: β-mercaptoethanol, proteinase K, 80% ethanol, RNase-free water, silanol magnetic beads (1 μm particle size), human BRAF / CCDC6-RET gene mutation detection reagent (fluorescent PCR method).

[0091] Experimental steps:

[0092] Sample lysis: Add the thyroid nodule puncture sample, 200 μL of lysis buffer A, 10 μL of β-mercaptoethanol, and 20 μL of proteinase K to a 1.5 mL centrifuge tube, vortex to mix, and incubate at 37°C for 15 minutes to allow the sample to fully lyse.

[0093] Binding treatment: Add 450 μL of binding buffer to the solution from the previous step, vortex mix for 2 minutes to fully expose the nucleic acid;

[0094] Magnetic bead binding: Add 20 μL of silanol magnetic beads to the solution, vortex mix for 5 minutes to fully bind the nucleic acid with the magnetic beads, then place the centrifuge tube on a magnetic rack for 1 minute and discard the supernatant;

[0095] Magnetic bead washing: Wash the magnetic beads three times with 80% ethanol, adding 500 μL of 80% ethanol each time. Vortex to mix and place on a magnetic rack for 1 minute. Discard the supernatant.

[0096] Nucleic acid elution: Add 50 μL of RNase-free water to the magnetic beads, vortex to mix, incubate at 37°C for 5 minutes, then place on a magnetic rack for 1 minute, and collect the elution buffer, which is the extracted DNA and RNA;

[0097] Fluorescent PCR detection: Using the human BRAF / CCDC6-RET gene mutation detection reagent, the extracted DNA and RNA were detected by fluorescent PCR according to the operation steps in the kit instructions, and the Ct values ​​were recorded.

[0098] Experimental results:

[0099] (I) BRAF gene mutation detection results:

[0100]

[0101]

[0102] (II) CCDC6-RET gene fusion detection results:

[0103]

[0104] Results Analysis: BRAF gene mutations were detected in all 22 BRAF gene-mutant thyroid nodule biopsy samples, with Ct values ​​ranging from 27.5 to 33.7. The internal control Ct value remained stable between 24.3 and 29.2, indicating that the extracted DNA quality was good and met the requirements for fluorescent PCR detection. No BRAF gene mutations were detected in the 3 wild-type BRAF samples, consistent with the sample type. CCDC6-RET gene fusion was detected in all 3 CCDC6-RET gene fusion samples, with Ct values ​​ranging from 22.1 to 25.0. The internal control Ct value ranged from 20.4 to 24.3, indicating that the extracted RNA had high integrity and purity, effectively supporting RNA-level gene fusion detection. These experimental results demonstrate that the method of this invention can efficiently extract DNA and RNA from clinical thyroid nodule biopsy samples, and the extracted nucleic acid quality is high, which can be directly used for downstream gene mutation and gene fusion detection, providing reliable technical support for the molecular diagnosis of thyroid tumors.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for simultaneously extracting DNA and RNA, characterized in that, The method includes: Sample lysis incubation: Incubate biological samples with lysis buffer, reducing agent and proteolytic enzymes; Add binding solution: Add binding solution to the mixture after pyrolysis incubation. The binding solution contains at least one liquid release agent, one nonionic surfactant and one alcohol. Nucleic acid binding solid phase: Utilizing the adsorption properties of silicon-containing solid phase materials for nucleic acids in a solution environment of pH 6-9, DNA and RNA are bound to a single solid phase; Washing and elution: After washing the solid phase with washing solution to remove impurities, DNA and RNA are eluted from the solid phase using elution solution.

2. The method for simultaneously extracting DNA and RNA according to claim 1, characterized in that, In the sample lysis incubation step, the lysis buffer contains at least one buffer compound and at least one anionic surfactant.

3. The method for simultaneously extracting DNA and RNA according to claim 2, characterized in that, The buffer compound is preferably selected from N-(tris(hydroxymethyl)methyl)glycine TRICINE, tris(hydroxymethyl)aminomethane TRIS, di(2-hydroxyethyl)iminotris(hydroxymethyl)methane BIS-TRIS, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) HEPES, N-cyclohexyl-2-aminoethanesulfonic acid CHES, N,N-bis(2-hydroxyethyl)glycine BICINE, 2-(N-morpholino)ethanesulfonic acid MES, piperazine-1,4-bis(2-ethanesulfonic acid) PIPES, 3-(N-morpholino)propanesulfonic acid MOPS, and phosphate buffers, and the buffer compound is present at a concentration ≤500 mM.

4. The method for simultaneously extracting DNA and RNA according to claim 2, characterized in that, The anionic surfactant is preferably a bile acid-based detergent, a sarcosine-based detergent, or a sulfate or sulfonate of a fatty alcohol, and is present at 0.1% to 20% w / v.

5. The method for simultaneously extracting DNA and RNA according to claim 1, characterized in that, The reducing agent is preferably selected from dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, dithioerythritol, sodium thiosulfate, β-mercaptoethanol, or a mixture thereof.

6. The method for simultaneously extracting DNA and RNA according to claim 1, characterized in that, The proteolytic enzyme is a protease, optionally a serine protease, present at a concentration ranging from 0.001 to 5% w / v, and the incubation temperature is set to 25–70°C, preferably 30–60°C.

7. The method for simultaneously extracting DNA and RNA according to claim 1, characterized in that, In the step of adding the binding liquid, the liquid release agent is preferably a sodium salt or guanidine salt liquid release agent, that is, a mixture of two or more of sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, and guanidine isothiocyanate.

8. The method for simultaneously extracting DNA and RNA according to claim 1, characterized in that, In the step of adding the binding liquid, the nonionic surfactant is preferably a polyoxyethylene-based nonionic detergent, including polyoxyethylene fatty acid esters and polyoxyethylene alkyl phenyl ethers, and the nonionic surfactant is present at a concentration in the range of 0.1% to 10% w / v.

9. The method for simultaneously extracting DNA and RNA according to claim 1, characterized in that, In the step of adding the binding liquid, the alcohol is preferably a branched or unbranched alcohol having 1-11 carbon atoms, most preferably selected from ethanol, ethylene glycol, isopropanol, n-butanol, and 1,3-butanediol, present at a concentration in the range of 1% to 75% w / v.

10. A method for simultaneously extracting DNA and RNA according to claim 1, characterized in that, In the washing and elution steps, the solid phase is washed 1 to 3 times with 80% ethanol to remove residual elution agent, protein and salt ions. During elution, an RNase-free water elution buffer is used to destroy the binding force between nucleic acids and the solid phase in a low-salt environment, thereby eluting DNA and RNA.

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