Lysate and method for extracting genome DNA

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a lysis solution and a method for extracting genome DNA (deoxyribonucleic acid). The lysate comprises 90 to 110 mM of Tris, 45 to 55 mM of EDTA (Ethylene Diamine Tetraacetic Acid), 90 to 110 mM of NaCl and 0.7 to 1.3 V% of SDS (Sodium Dodecyl Sulfate), and the pH (Potential of Hydrogen) of the lysate is 7.5 According to the novel wheat genome DNA extraction method provided by the invention, the subsequent experiment process is accelerated, and the working efficiency is improved. The average quality of the genome DNA extracted by the method is high, the concentration is greater than 1200 ng / mu L, and the subsequent experimental analysis requirements can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and specifically discloses a lysis solution and method for extracting genomic DNA. Background Art

[0002] Wheat is one of the most important food crops in China. Related molecular biology work is directly related to the safe production of food, and the extraction of high-quality genomic DNA is the basis of molecular biology research. To ensure the smooth progress of important technical links such as wheat germplasm resource identification, forward cloning of genes regulating important agronomic traits, and molecular marker-assisted breeding of genomic markers, it is very necessary to establish a method for rapidly and efficiently extracting wheat genomic DNA.

[0003] It is relatively difficult to isolate DNA from plant materials with strong heterogeneity such as wheat. At present, the extraction efficiency or quality of conventional CTAB method, SDS method, alkali treatment method, high-temperature water bath method, etc. is not satisfactory. The magnetic bead method and DNA kit extraction cannot meet the requirements of large-scale and low-cost, and are not suitable for the extraction and detection of a large number of wheat samples. Summary of the Invention

[0004] The present invention provides a lysis solution and method for extracting genomic DNA, develops a high-efficiency extraction process and corresponding reagents for wheat genomic DNA, and uses methods such as agarose gel electrophoresis and spectrophotometer to detect the integrity and purity of DNA. The genomic DNA has high purity and good integrity, meets various molecular experiments such as molecular marker detection and gene cloning, and the extraction steps are simplified, safe and efficient. It meets the requirements of large-scale, low-cost and high-quality genomic DNA extraction under general experimental conditions. This method is suitable for various different types of wheat and tissues, and the extracted genomic DNA has good quality and can meet the requirements of the next experiment. It is a general method for extracting wheat genomic DNA. At the same time, it is also applicable to the extraction of genomic DNA of plants such as corn, and has a wide range of applications.

[0005] The present invention provides a lysis solution, including: 90 - 110 mM Tris, 45 - 55 mM EDTA, 90 - 110 mM NaCl and 0.7 - 1.3 V% SDS, pH = 7.5 - 8.5. Using this lysis solution can accelerate the subsequent experimental process and improve work efficiency. The inventors used this lysis solution to extract genomic DNA with an average quality being relatively high, and the concentration being greater than 1200 ng / μL, which can meet the requirements of subsequent experimental analysis.

[0006] Preferably, it includes: 95 - 105 mM Tris, 48 - 52 mM EDTA, 95 - 105 mM NaCl and 0.8 - 1.2 V%% SDS, pH = 7.8 - 8.2.

[0007] More preferably, it includes 100 mM Tris, 50 mM EDTA, 100 mM NaCl, 1% V% SDS and pH = 8.

[0008] The present invention also provides a method for extracting DNA, using the lysis solution.

[0009] According to the method for extracting DNA, add the crushed sample to the lysis solution to lyse the tissue;

[0010] Preferably, if the sample is a fresh sample, the sample after adding the lysis solution is treated at 60 - 70 °C for 0.5 - 1 h, preferably 62 - 68 °C, and more preferably 65 °C;

[0011] Preferably, if the sample is a dry sample, the sample after adding the lysis solution is treated at room temperature for 0.5 - 1 h.

[0012] According to the method for extracting DNA, after lysing the tissue with the lysis solution, add potassium acetate to precipitate impurities, and discard the precipitate after centrifugation;

[0013] Preferably, the centrifugation is carried out at 2 - 6 °C, preferably 4 °C.

[0014] Preferably, the concentration of the potassium acetate solution is 5 M. After shaking evenly, it is necessary to perform high - speed centrifugation at low temperature to remove impurities such as proteins and reduce genomic DNA contamination.

[0015] According to the method for extracting DNA, add the supernatant obtained by centrifugation to isopropanol to extract genomic DNA;

[0016] Preferably, 0.5 - 0.7 times the volume of isopropanol is added to the supernatant, and more preferably 0.6 times the volume;

[0017] Preferably, the isopropanol is cold isopropanol; more preferably, it is isopropanol pre - cooled at - 22 to - 18 °C, and even more preferably pre - cooled at - 20 °C.

[0018] Preferably, the cold isopropanol is 0.6 times the volume of the supernatant and needs to be pre - frozen at - 20 °C in advance to improve the extraction effect of genomic DNA.

[0019] Preferably, after adding the cold isopropanol, let it stand at 4 °C for more than 20 min, which can significantly increase the amount of genomic DNA.

[0020] According to the method for extracting DNA, after extracting genomic DNA, let it stand and then centrifuge at room temperature and discard the supernatant.

[0021] According to the method for extracting DNA, wash the precipitate after centrifugation at room temperature with ethanol. Preferably, wash it 1 - 2 times with ethanol;

[0022] Preferably, elute with 70% ethanol, invert 3 - 5 times to fully wrap and elute the DNA precipitate with 70% ethanol. Its function is to remove isopropanol and reduce genomic DNA contamination.

[0023] Preferably, dry the product after ethanol elution at room temperature and then dissolve it in 1×TE or sterilized ddH2O;

[0024] Preferably, the TE includes 8 - 12 mM Tris and 0.8 - 1.2 mM EDTA, pH = 7.8 - 8.2;

[0025] More preferably, the TE includes 10 mM Tris and 1 mM EDTA, pH = 8.

[0026] According to the method for extracting DNA, the sample is a plant sample;

[0027] Preferably, the sample is wheat or corn tissue;

[0028] Preferably, the sample is a fresh sample or a dry sample;

[0029] More preferably, the fresh sample is a leaf or a shoot tip;

[0030] More preferably, the dry sample is a grain;

[0031] Preferably, if the sample is a fresh sample such as a leaf or a shoot tip, place the centrifuge tube in an oven, set the temperature to 65°C and process for 0.5 - 1 h; if the sample is dry grain, it can be left standing at room temperature for 0.5 - 1 h.

[0032] Preferably, it includes the following steps: using wheat or corn tissue as the treatment object, grinding and crushing it, adding a modified lysis solution to fully lyse the tissue, adding potassium acetate to precipitate impurities, centrifuging at low temperature and discarding the precipitate, adding 0.6 times the volume of cold isopropanol to the supernatant to extract genomic DNA, standing and then centrifuging at room temperature and discarding the supernatant, eluting with 70% ethanol 1 - 2 times, drying at room temperature and then dissolving it in 1×TE or sterilized ddH2O to obtain a high-quality wheat or corn genomic DNA solution; detecting the quality of the extracted genomic DNA.

[0033] According to the method for extracting DNA, use agarose gel electrophoresis to detect the quality of the genome;

[0034] Preferably, when using agarose gel to detect the quality of genomic DNA, the agarose gel is 1%, and the sample well contains an equal-volume mixture of genomic DNA dissolution solution and loading buffer (0.25% bromophenol blue and 40% sucrose).

[0035] Preferably, it includes using the primers shown in sequences 1 - 2 and / or sequences 3 - 4.

[0036] Preferably, the molar ratio of each single-stranded DNA in each primer pair is 1:1.

[0037] Preferably, the set of PCR reagents for detecting the quality of wheat genomic DNA is any one of the following 1) - 2):

[0038] 1) PCR reagents containing the primer pair Barc142;

[0039] 2) PCR reagents containing the primer pair Wmc28.

[0040] Preferably, the purity and concentration of genomic DNA are detected using a Nanodrop spectrophotometer. The control sample must be selected with the same DNA solvent. That is, when the solvent is 1×TE, the control is 1×TE; when the solvent is sterilized ddH2O, the control is sterilized ddH2O.

[0041] Preferably, the present invention provides a method for rapidly extracting DNA by lysing and treating tissues such as wheat or corn leaves and grains with SDS (sodium dodecyl sulfate), comprising the following steps: 1) Take 0.1 g of fresh plant samples from the field or greenhouse seedling trays and place them in a 2.0 mL centrifuge tube filled with steel beads; 2) After freeze-drying the centrifuge tube in liquid nitrogen for 1 min, take it out and place it in a grinder for rapid grinding for 45 s; 3) Add 1 mL of pre-prepared DNA extraction buffer (pH = 8, 100 mM Tris, 50 mM EDTA, 100 mM NaCl, 1% SDS) to the centrifuge tube and shake to mix evenly; 4) Place the centrifuge tube containing the sample and the extract in an oven, set the temperature to 65 °C and treat for 0.5 - 1 h; 5) Take out the treated sample, add 400 μL of 5 M potassium acetate solution to the centrifuge tube and shake to mix evenly; 6) Centrifuge at 12000 rpm for 10 min at 4 °C in a centrifuge; 7) Transfer 600 μL of the supernatant to a 1.5 ml centrifuge tube, add 360 μL of isopropanol pre-treated at -20 °C, mix well and let it stand at -20 °C for 20 min; 8) After centrifuging at 10000 rpm for 5 min, pour out the supernatant, and wash the precipitate with 70% ethanol 1 - 2 times; 9) Pour out the ethanol, dry the DNA at room temperature, and dissolve it with 200 μL of 1×TE or sterilized ddH2O.

[0042] The present invention also provides the application of the lysis solution or the method for extracting DNA in extracting genomic DNA from different types of wheat or corn tissues.

[0043] The beneficial effects of the present invention:

[0044] The present invention provides a new method for extracting genomic DNA, which can accelerate the subsequent experimental process and improve work efficiency. The inventors used this method to extract genomic DNA with an average high quality and a concentration greater than 1200 ng / μL, which can fully meet the requirements of subsequent experimental analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments.

[0046] Figure 1 It is a detection diagram of the integrity of the DNA sample extracted in Example 2 of the present invention;

[0047] Note: M, DNA maker; 1-6 are Zhongyu 9302, 04 Zhong 36, Bainong 64, Zhoumai 16, Zhoumai 22, and Nongda 399 in sequence.

[0048] Figure 2 It is a PCR amplification result diagram using SSR markers Barc142 and Wmc28 provided in Example 2 of the present invention;

[0049] M: DNA maker; 1: Zhongyu 9302; 2: 04 Zhong 36; 3: Bainong 64; 4, Zhoumai 16; 5: Zhoumai 22; 6: Nongda 399, 7: Zhongyu 9302; 8: 04 Zhong 36; 9: Bainong 64; 10: Zhoumai 16; 11: Zhoumai 22; 12: Nongda 399;

[0050] Note: 1-6 are amplified using Barc142; 7-12 are amplified using Wmc28.

[0051] Figure 3 It is a detection diagram of the integrity of the DNA sample extracted in Example 3 of the present invention;

[0052] Note: M, DNA maker; 1-6 are Zhongyu 9302, 04 Zhong 36, Bainong 64, Zhoumai 16, Zhoumai 22, and Nongda 399 in sequence.

[0053] Figure 4 It is a detection diagram of the integrity of the DNA sample extracted from maize in Example 4 of the present invention;

[0054] Note: M, DNA maker; 1 is B73, 2 is Mo17. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0055] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts fall within the scope of protection of the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0057] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained through commercial channels.

[0058] Zhongyu 9302: Independently selected and bred by the research group, with independent intellectual property rights, National Approval for Wheat Variety 20200062, Henan Approval for Wheat Variety 2014022.

[0059] 04 Zhong 36: Independently selected and bred by the research group, with independent intellectual property rights, Henan Approval for Wheat Variety 2006019.

[0060] Bainong 64: Selected and bred by Henan Institute of Science and Technology.

[0061] Zhoumai 16: Selected and bred by Zhoukou Academy of Agricultural Sciences, Henan Province, National Approval for Wheat Variety 2003029, Henan Approval for Wheat Variety 2002006.

[0062] Zhoumai 22: Selected and bred by Zhoukou Academy of Agricultural Sciences, Henan Province, National Approval for Wheat Variety 2007007.

[0063] Nongda 399: Selected and bred by China Agricultural University, Hebei Approval for Wheat Variety 20120004.

[0064] The preparation methods of DNA extraction solution (SDS), 5M potassium acetate (KAc), DNA dissolution solution (1×TE solution), 1% agarose gel, and 6% polyacrylamide gel in the following embodiments are as follows:

[0065] DNA extraction solution (SDS): 12.1 g of Tris, 14.6 g of EDTA, 5.85 g of sodium chloride (NaCl), 100 mL of 10% SDS (the solvent is water, and the solute and its concentration are SDS 100 g / L), adjust the pH value to 8.0, and add ddH2O to 1000 mL.

[0066] 5M potassium acetate (KAc): 490.7 g of potassium acetate, add ddH2O to 1000 mL.

[0067] DNA dissolution solution (1×TE solution): 1.21 g of Tris, 0.374 g of EDTA, adjust the pH value to 8.0, and add ddH2O to 1000 mL.

[0068] 1% Agarose Gel: 1 g of agarose is added to 100 mL of 1×TAE buffer (the solvent is water, and the solutes and their concentrations are Tris 4.84 g / L, EDTA 0.748 g / L). After mixing, it is boiled in a microwave oven at low temperature until dissolved. After it is not hot to the touch at room temperature, it is poured into a gel-casting tank with a comb inserted. After standing for 10 minutes, the comb is removed.

[0069] 6% Polyacrylamide Gel: 57 g of acrylamide, 3 g of bisacrylamide, 420 g of urea, 100 mL of 10×TBE buffer (the solvent is water, and the solutes and their concentrations are Tris 108 g / L, boric acid 55 g / L, EDTA 7.48 g / L), and ddH₂O is added to make up to 1000 mL.

[0070] The names of the SSR markers and their primer pairs in the following examples are the same. For example, the primer pair for the SSR marker Barc142 is Barc142.

[0071] Example 1. Obtaining Genomic DNA of Zhongyu 9302, 04 Zhong 36, Bainong 64, Zhoumai 16, Zhoumai 22, and Nongda 399 in Wheat

[0072] 1. Sample Collection and Preparation

[0073] Leaf Samples: Place steel beads in a labeled 2.0 mL centrifuge tube. Use scissors to cut 0.1 g of wheat leaves in a field or greenhouse seedling tray. Roll them into a ball and put them into the bottom of the centrifuge tube with forceps. Freeze-dry the centrifuge tube in liquid nitrogen for 1 min, then take it out and quickly grind it in a grinder for 45 s.

[0074] Grain Samples: Wrap 2 wheat grains with weighing paper and then with filter paper, crush them with a heavy object, and pour the particles into a labeled 2.0 mL centrifuge tube.

[0075] 2. Lysing Tissues with SDS Method DNA Extraction Buffer

[0076] Add 1 mL of pre-prepared DNA lysis solution (pH = 8, 100 mM Tris, 50 mM EDTA, 100 mM NaCl, 1% SDS) to the centrifuge tube containing the sample in Step 1, and shake and mix evenly. If the sample is a fresh sample such as leaves or shoot tips, place the centrifuge tube in an oven and set the temperature to 65°C for 0.5 - 1 h; if the sample is dry grains, it can be left standing at room temperature for 0.5 - 1 h.

[0077] 3. Removal of Impurities

[0078] Take out the sample after the treatment in Step 2, add 400 μL of 5 M potassium acetate solution into the centrifuge tube, mix well by shaking. SDS (sodium dodecyl sulfate) binds to proteins to form a complex and denatures and precipitates, releasing nucleic acids. At the same time, SDS reacts with potassium acetate to produce PDS (potassium dodecyl sulfate) precipitate. After centrifuging at 12,000 rpm for 10 min in a 4 °C centrifuge, discard the precipitate.

[0079] 4. Genomic DNA Extraction

[0080] Transfer 600 μL of the supernatant obtained in Step 3 to a labeled 1.5 mL centrifuge tube, add 360 μL of isopropanol that has been pre-treated at -20 °C, mix well, and let it stand at -20 °C for 20 min. After centrifuging at 10,000 rpm for 5 min at room temperature in a centrifuge, pour out the supernatant.

[0081] 5. Washing of Genomic DNA

[0082] Add 500 μL of 70% ethanol to the precipitate obtained in Step 4, invert it 3 - 5 times for elution, then pour out the eluate, and repeat the washing 1 - 2 times.

[0083] 6. Release of Genomic DNA

[0084] Pour out the 70% ethanol used for elution in Step 5. The residual ethanol in the tube can be removed by a vacuum pump. Dry the DNA at room temperature, and dissolve it with 200 μL of 1×TE or sterilized ddH2O.

[0085] Example 2. Detection of the Extraction Effect of Genomic DNA from Zhongyu 9302, 04 Zhong 36, Bainong 64, Zhoumai 16, Zhoumai 22, and Nongda 399 in Wheat

[0086] 1. Detection of the Quality of Genomic DNA by Agarose Gel Electrophoresis

[0087] Mix 2 μL of the genomic DNA dissolution solution obtained in Example 1 with 2 μL of loading buffer (0.25% bromophenol blue and 40% sucrose), and perform electrophoresis on a 1% agarose gel to detect the integrity (the electrophoresis buffer is 1×TAE).

[0088] The detection results are as Figure 1 shown. M: DNA maker; 1, Zhongyu 9302; 2, 04 Zhong 36; 3, Bainong 64; 4, Zhoumai 16; 5, Zhoumai 22; 6, Nongda 399.

[0089] 2. Detection of the Concentration and Purity of Genomic DNA by Nanodrop Spectrophotometer

[0090] Take 2 μL of genomic DNA lysate and use a Nanodrop spectrophotometer (ND-1000, Thermo Fisher Scientific, USA) to detect the concentration and purity of the DNA (OD 260 / OD 280 value).

[0091] Table 1. Concentrations and purities of genomic DNA from different samples

[0092]

[0093] Note: For relatively pure DNA, the OD 260 / OD 280 ratio should be around 1.8 - 2.0. A ratio lower than 1.8 indicates that proteins in the extracted DNA may not have been completely removed, and a ratio higher than 2.0 indicates that RNA may be present in the extracted DNA.

[0094] 3. PCR amplification detection of SSR markers Barc142 and Wmc28

[0095] Dilute the extracted genomic DNA 20-fold. Using the diluted DNA as a template, perform PCR amplification with wheat SSR primer pairs (Somers et al., 2004) that have relatively stable conventional PCR amplification results. In this invention, SSR markers Barc142 and Wmc28 on chromosome 5B are selected for PCR amplification to identify the quality of the extracted wheat genomic DNA. The sequences of the SSR marker primer pairs are shown in Table 2.

[0096] Table 2. Sequences of SSR markers Barc142 and Wmc28

[0097]

[0098]

[0099] Note: The product sizes in the table refer to the amplification results using most wheat genomic DNA as a template.

[0100] 1) Perform PCR amplification on the genomic DNA of the wheat sample to be tested using the primer pair of SSR marker Barc142 (composed of single-stranded DNAs shown in Sequence 1 and Sequence 2 of Table 2).

[0101] Reaction system: 1 μL of 10×PCR Buffer, 0.2 μL of 2.5 mM dNTPs, 0.2 μL of 10 mM forward primer F, 0.2 μL of 10 mM reverse primer R, 1 μL of DNA template (30 ng / μL), 0.1 μL of Taq DNA polymerase (5 U / μL, Taq DNA polymerase and the supporting Buffer and dNTPs were purchased from Baori Biotechnology (Beijing) Co., Ltd., RR001B), and made up to 10 μL with water.

[0102] Reaction procedure: Pre-denaturation at 95°C for 2 min; (denaturation at 94°C for 30 s; annealing at 55°C for 45 s; extension at 72°C for 30 s) for 32 cycles; extension at 72°C for 10 min.

[0103] The amplified products were detected by 6% polyacrylamide gel, and the quality of the wheat genomic DNA was judged according to the bands of the amplified products.

[0104] The detection results are as Figure 2 shown, M: DNA maker; 1, Zhongyu 9302; 2, 04 Zhong 36; 3, Bainong 64; 4, Zhoumai 16; 5, Zhoumai 22; 6, Nongda 399.

[0105] 2) PCR amplification was performed on the genomic DNA of the wheat to be tested using the primer pair of SSR marker Wmc28 (consisting of single-stranded DNAs shown in Sequence 3 and Sequence 4 of Table 2).

[0106] Reaction system: 1 μL of 10×PCR Buffer, 0.2 μL of 2.5 mM dNTPs, 0.2 μL of 10 mM forward primer F, 0.2 μL of 10 mM reverse primer R, 1 μL of DNA template (30 ng / μL), 0.1 μL of Taq DNA polymerase (5 U / μL, Taq DNA polymerase and the supporting Buffer were purchased from Baori Biotechnology (Beijing) Co., Ltd., RR001B), and made up to 10 μL with water.

[0107] Reaction procedure: Pre-denaturation at 95°C for 2 min; (denaturation at 94°C for 30 s; annealing at 55°C for 45 s; extension at 72°C for 30 s) for 32 cycles; extension at 72°C for 10 min.

[0108] The amplified products were detected by 6% polyacrylamide gel, and the quality of the wheat genomic DNA was judged according to the bands of the amplified products.

[0109] The detection results are as Figure 2 shown, M: DNA maker; 7, Zhongyu 9302; 8, 04 Zhong 36; 9, Bainong 64; 10, Zhoumai 16; 11, Zhoumai 22; 12, Nongda 399.

[0110] 4. Result Analysis

[0111] Figure 1 , Table 1 and Figure 2 The results show that:

[0112] In Step 1, detected by 1% agarose gel electrophoresis, Figure 1 the results show that the integrity of all wheat genomic DNAs is good and there is almost no degradation;

[0113] In Step 2, after extracting genomic DNA by this method, the concentration and purity were detected by Nanodrop spectrophotometer. The concentration of all genomic DNAs was above 1200 ng / μL, and the OD 260 / OD 280 values were all between 1.8 and 2.0, with low pollution degree and relatively uniform purity;

[0114] In Step 3, in Step 1), the primer pair Barc142 was used to perform PCR amplification on the genomic DNA of the wheat to be tested. The amplification bands of all genomic DNAs were relatively bright, proving that the quality of the extracted genomic DNA was good and it was suitable for subsequent molecular biology research;

[0115] In Step 3, in Step 2), the primer pair Wmc28 was used to perform PCR amplification on the genomic DNA of the wheat to be tested. The amplification bands of all genomic DNAs were relatively bright, proving that the quality of the extracted genomic DNA was good and it was suitable for subsequent molecular biology research.

[0116] Example 3. Obtaining Genomic DNAs of Zhongyu 9302, 04 Zhong 36, Bainong 64, Zhoumai 16, Zhoumai 22, and Nongda 399 in Wheat

[0117] The difference from Example 1 is that the concentration of SDS in the DNA lysis solution was 0.8%, and wheat genomic DNA was obtained. The integrity of the obtained wheat genomic DNA samples was detected by the method of Example 2, as Figure 3 shown.

[0118] Example 4. Obtaining Genomic DNAs of Maize B73 and Mo17

[0119] The difference from Example 1 is that fresh maize leaves were collected to extract maize genomic DNA. The integrity of the obtained maize genomic DNA samples was detected, as Figure 4 shown.

[0120] The above results show that the rapid genomic DNA extraction method provided by the present invention obtains DNA with high purity and good integrity, meets various molecular experiments such as molecular marker detection and gene cloning, simplifies the extraction steps, is safe and efficient, and is a general and efficient genomic DNA extraction method.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lysis solution, characterized in that, Comprising: 90 - 110 mM Tris, 45 - 55 mM EDTA, 90 - 110 mM NaCl and 0.7 - 1.3 V% SDS, pH = 7.5 - 8.5; Preferably, comprising: 95 - 105 mM Tris, 48 - 52 mM EDTA, 95 - 105 mM NaCl and 0.8 - 1.2 V%% SDS, pH = 7.8 - 8.2; More preferably, comprising 100 mM Tris, 50 mM EDTA, 100 mM NaCl, 1% V% SDS and pH = 8.

2. A method for extracting DNA, characterized in that, Using the lysis solution described in claim 1.

3. The method for extracting DNA according to claim 2, characterized in that, Adding the pulverized sample to the lysis solution to lyse the tissue; Preferably, if the sample is a fresh sample, the sample after adding the lysis solution is treated at 60 - 70 °C for 0.5 - 1 h, preferably 62 - 68 °C, more preferably 65 °C; Preferably, if the sample is a dry sample, the sample after adding the lysis solution is treated at room temperature for 0.5 - 1 h.

4. The method for extracting DNA according to claim 2 or 3, characterized in that, After lysing the tissue with the lysis solution, adding potassium acetate to precipitate impurities, and discarding the precipitate after centrifugation; Preferably, the centrifugation is carried out at 2 - 6 °C, preferably 4 °C.

5. The method for extracting DNA according to claim 4, characterized in that, Adding the supernatant obtained by centrifugation to isopropanol to extract genomic DNA; Preferably, 0.5 - 0.7 times the volume of isopropanol is added to the supernatant, more preferably 0.6 times the volume; Preferably, the isopropanol is cold isopropanol; more preferably, it is isopropanol pre-cooled at -22 to -18 °C, even more preferably pre-cooled at -20 °C.

6. The method for extracting DNA according to claim 5, characterized in that, After extracting genomic DNA, let it stand and then discard the supernatant after centrifugation at room temperature.

7. The method for extracting DNA according to claim 6, characterized in that, Washing the precipitate after centrifugation at room temperature with ethanol, preferably, washing with ethanol 1 - 2 times; Preferably, the product after ethanol washing is dried at room temperature and then dissolved in 1×TE or sterilized ddH2O; Preferably, the TE comprises 8 - 12 mM Tris and 0.8 - 1.2 mM EDTA, pH = 7.8 - 8.2; More preferably, the TE comprises 10 mM Tris and 1 mM EDTA, pH = 8.

8. The method for extracting DNA according to any one of claims 2 to 7, characterized in that, The sample is a plant sample; Preferably, the sample is wheat or corn tissue; Preferably, the sample is a fresh sample or a dry sample; More preferably, the fresh sample is a leaf or a shoot tip; More preferably, the dry sample is a grain; Preferably, it comprises the following steps: using wheat or corn tissue as the treatment object, grinding and pulverizing it, adding the improved lysis solution to fully lyse the tissue, then adding potassium acetate to precipitate impurities, discarding the precipitate after freezing centrifugation, adding 0.6 times the volume of cold isopropanol to the supernatant to extract genomic DNA, letting it stand and then discarding the supernatant after centrifugation at room temperature, washing with 70% ethanol 1 - 2 times, drying at room temperature and then dissolving in 1×TE or sterilized ddH2O to obtain a high-quality wheat or corn genomic DNA solution; detecting the quality of the extracted genomic DNA.

9. The method for extracting DNA according to any one of claims 2 to 8, characterized in that, Detecting the quality of the genome using agarose gel electrophoresis; Preferably, it comprises using the primers shown in sequences 1 - 2 and / or sequences 3 - 4.

10. The lysis solution according to claim 1 or the method for extracting DNA according to any one of claims 2 to 9 is applied to extract genomic DNA of different types of wheat or corn tissues.

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