High-yield animal muscle tissue mitochondrial DNA extraction method

Abstract

The invention relates to the technical field of molecular biology, particularly discloses a high-yield animal muscle tissue mitochondrial DNA extraction method, and aims to solve the technical problems of insufficient mitochondrial release, low DNA yield and insufficient concentration when an existing extraction method is applied to animal muscle tissues. According to the method, the effect of improving the mitochondrial DNA yield is achieved through the steps of sample homogenization, mitochondrial separation, mitochondrial splitting decomposition, mixing treatment, DNA precipitation, DNA washing, DNA dissolution and the like. The concentration of the mitochondrial DNA extracted by the method is increased by 7-9 times compared with that of a commercial kit, and the DNA is good in integrity, high in purity and free of obvious protein pollution or degradation. The method is simple and convenient to operate, the reagent is easy to prepare, the repeatability is high, the downstream experiment requirements of high-sensitivity PCR amplification, gene sequencing and the like can be met, and the method has remarkable practical value.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to a method for high-yield extraction of mitochondrial DNA from animal muscle tissue. Background Technology

[0002] Mitochondrial DNA, as a crucial genetic material in animal cells, plays an irreplaceable role in research on animal genetics, evolutionary biology, and species identification. Currently, extracting mitochondrial DNA from animal tissues using commercial mitochondrial DNA extraction kits has become a common laboratory technique, widely applied in various related experiments. However, existing commercial kits still have certain limitations when extracting mitochondrial DNA from animal muscle tissue.

[0003] Animal muscle tissue, rich in muscle fibers and connective tissue, has a relatively dense structure. Following standard kit procedures often fails to fully release mitochondria, resulting in low concentrations and yields of extracted mitochondrial DNA. Besides commercial kits, traditional extraction methods such as the phenol-chloroform method and salting-out method suffer from complex procedures, easy DNA degradation, and low yields in dense muscle tissue. Furthermore, some kits and other commonly used methods have limited DNA capture efficiency during the precipitation step, further affecting the final extraction results and failing to meet the demands of subsequent experiments such as high-sensitivity PCR amplification and gene sequencing for high-concentration, high-yield mitochondrial DNA.

[0004] More importantly, for specialized samples like muscle tissue, existing kits lack optimized operating parameters (such as sample volume and reagent ratios), which can easily lead to DNA degradation during extraction, thus affecting the accuracy and reliability of subsequent experimental results. Therefore, a method specifically designed for animal muscle tissue to effectively extract mitochondrial DNA is needed to improve its concentration and yield, which has significant practical application value. Summary of the Invention

[0005] This invention aims to address the problems of low yield and insufficient concentration in existing mitochondrial DNA extraction kits when extracting mitochondrial DNA from animal tissues (especially muscle tissue). The standard operating procedures of existing kits are not adequately adapted to animal muscle tissue. Because muscle tissue is rich in muscle fibers and connective tissue, and has a dense structure, it is difficult to fully release mitochondria using conventional steps. Furthermore, the DNA precipitation efficiency is limited, resulting in low concentrations and yields of extracted mitochondrial DNA, which cannot meet the needs of downstream experiments such as high-sensitivity PCR amplification and gene sequencing.

[0006] To address the aforementioned technical problems, this invention provides a method for high-yield mitochondrial DNA extraction from animal tissues, comprising the following steps:

[0007] (1) Sample preparation: Take animal muscle tissue, remove impurities, rinse, absorb surface moisture, cut into small pieces, and weigh no less than 1g of tissue sample;

[0008] (2) Sample homogenization: The tissue sample was transferred to a homogenization tube, placed on ice, and homogenization buffer was added for homogenization to obtain a homogenate.

[0009] (3) Mitochondrial isolation: The homogenate was centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant was collected. The supernatant was then centrifuged at 12000-14000 rpm for 25-30 minutes, and the supernatant was discarded to obtain mitochondrial precipitate from animal muscle tissue.

[0010] (4) Mitochondrial lysis: Add buffer A to the mitochondrial precipitate of animal muscle tissue for resuspension, then add buffer B, mix until completely suspended, and incubate at 55°C for 60 minutes, inverting and mixing 1-2 times every 5 minutes during incubation.

[0011] (5) Mixing treatment: Add buffer C to the lysed mixture and mix well;

[0012] (6) DNA precipitation: Centrifuge the mixed solution at 4°C, collect the supernatant, add twice the volume of precipitation reagent to the supernatant, and add one-tenth the volume of sodium acetate solution of precipitation reagent. Mix well and let stand at -20°C, then centrifuge again, discard the supernatant, and obtain DNA precipitation from animal muscle tissue.

[0013] (7) DNA washing: Add buffer D to the DNA precipitate of the animal muscle tissue, vortex until the DNA precipitate is suspended, centrifuge at 4°C and discard the supernatant;

[0014] (8) DNA dissolution: The washed DNA precipitate was dried at 20℃~25℃ and dissolved in dissolution buffer to obtain animal tissue mitochondrial DNA solution.

[0015] Preferably, the homogenization buffer contains 0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA·2Na and 0.1% (w / v) BSA, with a pH of 7.5.

[0016] Preferably, the buffer solution A contains 0.5 M NaCl, 10 mM Tris-HCl and 1 mM EDTA·2Na, and has a pH of 7.5.

[0017] Preferably, the buffer solution B contains 3 M NaCl, 10 mM Tris-HCl and 1 mM EDTA·2Na, and has a pH of 7.5.

[0018] Preferably, the buffer solution C is a solution containing sodium dodecyl sulfate, and the concentration of sodium dodecyl sulfate is 0.1 g / mL.

[0019] Preferably, the buffer solution D contains 10 mM Tris-HCl, 0.1 M NaCl, and ethanol at a final concentration of 70% by volume, with a pH of 7.5.

[0020] Preferably, the dissolution buffer contains 10 mM Tris-HCl and 1 mM EDTA·2Na, with a pH of 7.5.

[0021] Preferably, in step (6), the precipitating agent is ethanol, and the concentration of the sodium acetate solution is 2 mol / L.

[0022] Preferably, the centrifugation conditions for the supernatant in step (3) are 12,500 rpm for 25 minutes.

[0023] Preferably, for every 1g of tissue sample, the amount of homogenization buffer added is 2 mL, the amount of buffer A added is 500 μL, the amount of buffer B added is 80 μL, the amount of buffer C added is 200 μL, the amount of buffer D added is 0.5 mL, and the amount of dissolution buffer added is 50 μL.

[0024] The technical solution of this invention was derived through extensive experiments, comparisons, and analyses. By increasing the sample size and optimizing the homogenization conditions, mitochondria in muscle tissue are released more fully, effectively reducing insufficient mitochondrial release due to dense tissue structure, thereby minimizing DNA loss during extraction. In the DNA precipitation step, a combined precipitation system of sodium acetate and ethanol is used, effectively improving the precipitation efficiency and yield of DNA. Experimental results show that under the optimized conditions, the concentration of extracted mitochondrial DNA is approximately 7-9 times higher than under unoptimized conditions. Experimental analysis determined that centrifugation at 12500 rpm for 25 minutes is the optimal mitochondrial separation parameter. This condition ensures sufficient mitochondrial precipitation while avoiding co-precipitation of impurities due to excessively high centrifugation speed or mitochondrial loss due to excessively low speed, thus facilitating the smooth progress of subsequent lysis steps. Furthermore, the mitochondrial DNA obtained by the method of this invention exhibits good integrity and high stability, maintaining good preservation at -20℃ or 4℃. Compared with common commercial kits and crude extraction methods such as modified salting-out, this invention has significant advantages in extraction efficiency, DNA concentration, and integrity. Therefore, this invention effectively improves the extraction efficiency and quality of mitochondrial DNA from animal muscle tissue, and has good application value and prospects. Attached Figure Description

[0025] Figure 1Mitochondrial precipitate diagram, where: 1 is the mitochondrial precipitate obtained with the commonly recommended sample amount for mitochondrial DNA extraction, and 2 is the mitochondrial precipitate obtained with an increased sample amount; No. 1 is the mitochondrial precipitate obtained when extracting with the recommended sample amount (200 mg) of the commercial kit, the amount of precipitate is small, indicating that the mitochondria are not fully released under the conventional sample amount; No. 2 is the mitochondrial precipitate obtained by extraction using the method of this invention, the amount of precipitate is significantly greater than No. 1, indicating that increasing the sample amount can effectively improve the release amount and precipitation efficiency of mitochondria, laying the foundation for a high yield of subsequent DNA extraction.

[0026] Figure 2 The results of gel electrophoresis of mitochondrial DNA in chicken tissue (① and ② are parallel experiments under this method), where: M is the DNA Ladder band, 1 is the improved salting-out method, 2 is the method of commercial kit, 3 is method one of this invention (control method), and 4 is method two of this invention (method of this invention).

[0027] Figure 3 The PCR gel electrophoresis results of mitochondrial DNA from chicken tissue (① and ② are parallel experiments under this method), where: M is the DNA ladder band, 1 is the modified salting-out method, 2 is the commercial kit method, 3 is method one of this method, and 4 is method two of this method.

[0028] Figure 4 The results of mitochondrial DNA gel electrophoresis in pork tissue (① and ② are parallel experiments under this method), where: M is the DNA ladder band, 1 is the modified salting-out method, 2 is the commercial kit method, 3 is method one of this method, and 4 is method two of this method.

[0029] Figure 5 The PCR gel electrophoresis results of mitochondrial DNA from pork tissue are shown in the figure (① and ② are parallel experiments under this method). In the figure, M is the DNA ladder band, 1 is the modified salting out method, 2 is the commercial kit method, 3 is method one of this method, and 4 is method two of this method. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] The primers used in the following examples were prepared by Suzhou Genewiz Biotechnology Co., Ltd.

[0034] In the following examples, PCR amplification was performed using 2×Tag Master Mix (Dye Plus), which was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0035] It should be noted that all the following reagents were prepared independently:

[0036] The homogenization buffer consisted of: 0.25 M sucrose, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA·2Na, and 0.1% (w / v) BSA.

[0037] Weigh out sucrose, Tris, and EDTA·2Na according to the set concentration. Add sucrose to part of deionized water and stir until completely dissolved. Add Tris and EDTA·2Na in sequence and stir until completely dissolved. Then add BSA and stir gently until dissolved, avoiding vigorous stirring to prevent foaming. Adjust the pH of the solution to 7.5 with HCl. After making up to volume, autoclave at 121℃ for 20 min to obtain the homogenate buffer, and store at 4℃ for later use.

[0038] Buffer A consists of: 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA·2Na.

[0039] Weigh out the appropriate amount of NaCl according to the set concentration, add it to an appropriate amount of deionized water, and stir until completely dissolved; then add the appropriate amounts of Tris and EDTA·2Na in sequence, and stir until completely dissolved; adjust the pH of the solution system to 7.5 with HCl, make up to volume and sterilize to obtain buffer A, which is stored at 4℃ for later use.

[0040] Buffer B consists of 3 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA·2Na. Weigh the corresponding mass of NaCl according to the set concentration, add it to an appropriate amount of deionized water, and stir until completely dissolved. Then add the corresponding masses of Tris and EDTA·2Na in sequence, and stir until completely dissolved. Adjust the pH of the solution to 7.5 with HCl, bring the volume to a final volume, sterilize, and store at 4°C for later use.

[0041] Buffer C consists of 10% SDS (sodium dodecyl sulfate). Weigh 10 g of SDS according to the set concentration and dissolve it in 80 mL of deionized water. Heat at 50-60℃ and stir until completely dissolved; bring the volume to 100 mL and store at room temperature.

[0042] Buffer D consists of: 10 mM Tris-HCl (pH 7.5), 0.1 M NaCl, and 70% ethanol (by volume). Before use, add anhydrous ethanol at a volume ratio of 3:7, mix well, and store at 4°C.

[0043] The dissolution buffer consists of 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA·2Na. Weigh the appropriate amounts of Tris-HCl and EDTA·2Na according to the set concentration, and add them sequentially to deionized water, stirring until completely dissolved. Adjust the pH of the solution to 7.5 with HCl, bring the volume to a final volume, sterilize, and store at 4°C for later use.

[0044] Example 1: Extraction of mitochondrial DNA from chicken tissue

[0045] (1) Sample preparation: Select fresh chicken muscle tissue, remove surface impurities, rinse with physiological saline 2-3 times, absorb surface moisture with filter paper, cut the muscle tissue into small pieces of 1-2 mm³, and weigh 1 g of tissue sample.

[0046] (2) Sample homogenization: Place the processed sample into a homogenization tube, place it on ice, add 2 mL of homogenization buffer, and use a tissue homogenizer to homogenize until a uniform tissue homogenate is formed. During homogenization, pause for 5 seconds every 15 seconds to avoid over-homogenization and damage to the mitochondrial structure.

[0047] (3) Mitochondrial isolation: Transfer the homogenate to a centrifuge tube and centrifuge at 1000 rpm for 10 minutes at 4°C. Take the supernatant into a new centrifuge tube and centrifuge at 12500 rpm for 25 minutes at 4°C. Discard the supernatant and retain the mitochondrial precipitate.

[0048] (4) Mitochondrial lysis: Add 500 μL of Buffer A to the mitochondrial pellet to resuspend, then add 80 μL of Buffer B. After complete resuscitation by pipetting, incubate at 55°C for 60 minutes. During this period, gently invert the centrifuge tube 1-2 times every 5 minutes to ensure that the mitochondria are fully lysed.

[0049] (5) Mixing treatment: Add 200 μL of Buffer C to the lysed mixture and invert at least 30 times until fully mixed;

[0050] (6) DNA precipitation: Centrifuge the mixed solution at 12500 rpm for 20 minutes at 4℃, collect the supernatant into a new 2 mL EP tube, add 2 volumes of ethanol as precipitation reagent, then add 1 / 10 volume of 2 M sodium acetate solution, invert and mix 5-8 times, and let stand at -20℃ for 10 minutes; then centrifuge at 12000 rpm for 15 minutes, discard the supernatant, and retain the DNA precipitate;

[0051] (7) DNA washing: Add 0.5 mL of Buffer D to the DNA precipitate, vortex to suspend the precipitate, centrifuge at 12000 rpm for 5 minutes at 4℃, discard the supernatant, and repeat the washing once.

[0052] (8) DNA dissolution: After washing, the DNA precipitate was air-dried at room temperature to avoid excessive drying. 50 μL of dissolution buffer was added and the mixture was pipetted until completely dissolved to obtain the animal tissue mitochondrial DNA solution.

[0053] (9) DNA quality inspection: DNA is visually detected by agarose gel electrophoresis, and DNA concentration and purity are detected by ultra-micro spectrophotometer.

[0054] The extraction method described in this embodiment was compared with the improved salting-out method and the extraction method of commercial reagent kits, and the following Table 1 was obtained: DNA concentration and purity results detected by ultra-micro spectrophotometer.

[0055] In Table 1, number 1 represents the improved salting-out method, which is complex to operate and may result in protein contamination or degradation; number 2 represents the method using a commercial kit, which has a low mitochondrial DNA yield; number 3 represents Method 1 of this invention (the control method, which uses the same sample volume, homogenization conditions, centrifugation conditions, and other steps and experimental conditions as the method of this invention, but does not use sodium acetate to assist precipitation in the DNA precipitation step, and only uses ethanol precipitation), which significantly increases the concentration; and number 4 represents Method 2 of this invention (the method described in Example 1 of this invention, which increases the sample volume, optimizes the homogenization and centrifugation conditions, and uses sodium acetate to assist precipitation), which has a high DNA yield.

[0056] Table 1:

[0057]

[0058] Note: Method 1 refers to the absence of sodium acetate-assisted precipitation; Method 2 refers to increasing the sample size, optimizing homogenization and centrifugation conditions, and using sodium acetate-assisted precipitation.

[0059] Example 2: Gel electrophoresis detection of chicken mitochondrial DNA

[0060] This embodiment verifies the quality of the mitochondrial DNA extracted in Example 1 by directly performing gel electrophoresis after extraction. The verification steps are as follows:

[0061] To prepare a 1% agarose gel: Weigh 0.6 g of agarose, add 60 mL of 1×TAE buffer, heat until completely dissolved, cool to 50-60℃, add 6 μL of nucleic acid staining solution, mix well, pour into a gel casting plate, insert a comb, and wait for the gel to solidify.

[0062] Sample preparation: Take 9 μL of extracted chicken muscle tissue mitochondrial DNA solution and mix it with 1 μL of 10× DNA loading buffer.

[0063] Sample loading and electrophoresis: Add the mixed sample to the gel wells, and add 8 μL of DNA molecular weight standard (such as DL15000 bp). Electrophore in 1×TAE buffer at 120V for 30 minutes.

[0064] Imaging: After electrophoresis, the gel was placed in a gel imaging system for observation and photographing to obtain the gel electrophoresis results of mitochondrial DNA from chicken tissue, as shown in the image. Figure 2 As shown.

[0065] This embodiment uses agarose gel electrophoresis to detect the quality of chicken mitochondrial DNA. The results show that: the improved salting-out method resulted in severe band tailing, indicating protein contamination or DNA degradation; although the concentration was high, the quality was poor; the commercial kit method showed weak band brightness and extremely low yield; Method 1 of this invention showed approximately 7 times higher band brightness compared to the kit method, and was clear without tailing; Method 2 of this invention showed the brightest band brightness. It is evident that the method of this invention significantly improves the yield of mitochondrial DNA while maintaining DNA integrity.

[0066] Example 3: PCR verification of the quality of mitochondrial DNA from chicken tissue

[0067] This embodiment verifies the quality of the mitochondrial DNA extracted in Example 1 using PCR amplification. The verification steps are as follows:

[0068] PCR experiments were performed using extracted DNA as a template: The PCR amplification reaction system was 50 μL: DNA template (1 μg), 2×Taq PCR Mix 25 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, and ddH2O was added to bring the total volume to 50 μL; the forward primer for the chicken species was 5'-CTATAATCGATAATCCACGATTCA-3'; the reverse primer was 5'-CTTGACCTGTCTTATTAGCGAGG-3'; the amplification length was 131 bp;

[0069] The amplification conditions were: 94℃ for 3 min, 94℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, 35 cycles, 72℃ for 5 min, and the reaction was terminated. The amplification product was stored in a refrigerator at 4℃.

[0070] To prepare a 2% agarose gel: Weigh 1.2 g of agarose, add 60 mL of 1×TAE buffer, heat until completely dissolved, cool to 50-60℃, add 6 μL of nucleic acid staining solution, mix well, pour into a gel casting plate, insert a comb, and wait for the gel to solidify.

[0071] Sample loading and electrophoresis: Add 10 μL of amplified sample to the gel well, and add 8 μL of DNA molecular weight standard (such as DL2000) at the same time. Electrophore in 1×TAE buffer at 120V for 30 minutes.

[0072] Imaging: After electrophoresis, the gel was placed in a gel imaging system for observation and photographing to obtain a gel image of chicken tissue mitochondrial DNA PCR, as shown below. Figure 3 As shown.

[0073] In this embodiment, extracted chicken mitochondrial DNA was used as a template for 131 bp specific PCR amplification and electrophoresis detection. The results showed that: the improved salting-out method resulted in slightly tailed bands and poor amplification effect; the commercial kit method resulted in clear but low-brightness bands, indicating insufficient template concentration; the method of the present invention (Method 1) showed band brightness higher than 2, significantly higher and with consistent clarity; the method of the present invention (Method 2) showed the brightest bands and the strongest PCR amplification signal. This demonstrates that the DNA extracted by the present invention has high purity and sufficient concentration, meeting the requirements of downstream PCR amplification experiments.

[0074] Example 4: Extraction of mitochondrial DNA from pork tissue

[0075] Mitochondrial DNA was extracted from porcine muscle tissue using the general extraction steps 1-8 in Example 1 above, with a sample size of 1 g. This example also compared the improved salting-out method with a commercial kit extraction method, yielding the following results for DNA concentration and purity detected by an ultra-micro spectrophotometer. In Table 2, number 1 represents the improved salting-out method, which is complex and may result in protein contamination or degradation; 2 represents the kit method, resulting in low mitochondrial DNA yield; 3 represents Method 1 (the control method, as described in Table 1 above), showing a significant increase in concentration; and 4 represents Method 2 (the method described in Example 1 of this invention, as described in Table 1 above), resulting in a higher DNA yield.

[0076] Table 2:

[0077]

[0078] Note: Method 1 refers to the absence of sodium acetate-assisted precipitation; Method 2 refers to increasing the sample size, optimizing homogenization and centrifugation conditions, and using sodium acetate-assisted precipitation.

[0079] Example 5: Gel electrophoresis detection of mitochondrial DNA in pork

[0080] The method and steps are the same as in Example 2.

[0081] like Figure 4 In this embodiment, the detection of pork mitochondrial DNA by agarose gel electrophoresis showed that: the improved salting-out method resulted in severe band tailing, indicating significant contamination and degradation; the commercial kit method resulted in extremely weak band brightness and very low yield; the method of the present invention showed significantly higher and clearer band brightness without tailing, and the method of the second method showed the brightest brightness, with good consistency of bands in parallel experiments.

[0082] Example 6: PCR verification of the quality of mitochondrial DNA from pork tissue

[0083] The procedure was performed using the steps described in Example 3 above. The forward primer for the pig species was 5'-GCCTAAATCTCCCCTCAATGGTA-3'; the reverse primer was 5'-ATAAAAGAGGCAAATAGATTTTCG-3'; and the amplification length was 212 bp. Figure 5 As stated above.

[0084] In this embodiment, 212 bp specific PCR amplification was performed using extracted pork mitochondrial DNA as a template, followed by electrophoresis detection. The results showed that the improved salting-out method resulted in band tailing and poor amplification effect; the commercial kit method resulted in clear bands but low brightness and insufficient template concentration; Method 1 showed significantly higher band brightness (higher than 2) and consistent clarity; Method 2 had the brightest brightness and the strongest PCR amplification signal.

Claims

1. A method for high-yield extraction of mitochondrial DNA from animal muscle tissue, characterized in that, Includes the following steps: (1) Sample preparation: Take animal muscle tissue, remove impurities, rinse, absorb surface moisture, cut into small pieces, and weigh no less than 1g of tissue sample; (2) Sample homogenization: The tissue sample was transferred to a homogenization tube, placed on ice, and homogenization buffer was added for homogenization to obtain a homogenate. (3) Mitochondrial isolation: The homogenate was centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant was collected. The supernatant was then centrifuged at 12000-14000 rpm for 25-30 minutes, and the supernatant was discarded to obtain mitochondrial precipitate of animal muscle tissue. (4) Mitochondrial lysis: Add buffer A to the mitochondrial precipitate of the animal muscle tissue to resuspend, then add buffer B, mix until completely suspended, and incubate at 55°C for 60 minutes, inverting and mixing 1-2 times every 5 minutes during incubation. (5) Mixing treatment: Add buffer C to the lysis buffer obtained in step (4) and mix well; (6) DNA precipitation: Centrifuge the mixed solution at 4°C, collect the supernatant, add twice the volume of precipitation reagent to the supernatant, and add one-tenth the volume of sodium acetate solution of precipitation reagent. Mix well and let stand at -20°C, then centrifuge again, discard the supernatant, and obtain DNA precipitation from animal muscle tissue. (7) DNA washing: Add buffer D to the DNA precipitate of the animal muscle tissue, vortex until the DNA precipitate is suspended, centrifuge at 4°C, discard the supernatant, and obtain the DNA precipitate; (8) DNA dissolution: The DNA precipitate was dried at 20℃~25℃ and dissolved in dissolution buffer to obtain animal tissue mitochondrial DNA solution.

2. The method according to claim 1, characterized in that, The homogenization buffer contains 0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA·2Na and 0.1% (w / v) BSA, with a pH of 7.

5.

3. The method according to claim 2, characterized in that, The buffer solution A contains 0.5 M NaCl, 10 mM Tris-HCl and 1 mM EDTA·2Na, with a pH of 7.

5.

4. The method according to claim 3, characterized in that, The buffer solution B contains 3 M NaCl, 10 mM Tris-HCl and 1 mM EDTA·2Na, with a pH of 7.

5.

5. The method according to claim 4, characterized in that, The buffer solution C is a solution containing sodium dodecyl sulfate, and the concentration of sodium dodecyl sulfate is 0.1 g / mL.

6. The method according to claim 5, characterized in that, The buffer solution D contains 10 mM Tris-HCl, 0.1 M NaCl, and 70% volume ethanol at a final concentration, with a pH of 7.

5.

7. The method according to claim 6, characterized in that, The dissolution buffer contains 10 mM Tris-HCl and 1 mM EDTA·2Na, with a pH of 7.

5.

8. The method according to claim 7, characterized in that, In step (6), the precipitating agent is ethanol, and the concentration of the sodium acetate solution is 2 mol / L.

9. The method according to claim 8, characterized in that, In step (3), the supernatant is centrifuged at 12,500 rpm for 25 minutes.

10. The method according to claim 9, characterized in that, For every 1g of tissue sample, the amount of homogenization buffer added is 2 mL, the amount of buffer A added is 500 μL, the amount of buffer B added is 80 μL, the amount of buffer C added is 200 μL, the amount of buffer D added is 0.5 mL, and the amount of dissolution buffer added is 50 μL.

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