Kit for extracting plant genome DNA by paramagnetic particle method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a kit for extracting plant genome DNA through a paramagnetic particle method and application of the kit. The invention provides the binding liquid, the binding liquid can be used for adsorbing DNA by magnetic beads in the process of extracting genome DNA by a magnetic bead method, the concentration and the purity of an extraction product obtained in the genome DNA extraction process can be improved, and a better extraction effect is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a kit for extracting plant genomic DNA using a magnetic bead method and application thereof. Background Art

[0002] DNA, as a biological macromolecule that carries the genetic information necessary for synthesizing RNA and proteins, is the basis for related research. Therefore, it is particularly important to extract high-purity, high-yield, and high-integrity genomic DNA efficiently and quickly. However, plant tissue samples are complex and diverse. Unlike animal tissues, plant cells not only have cell walls, but some samples also contain a large amount of polysaccharides, oils, phenolic substances, and pigments. These secondary metabolites seriously affect the quality of plant genomic DNA extraction.

[0003] At present, the traditional plant genomic DNA extraction method and magnetic bead method are commonly used in laboratories. Among them, the traditional plant genomic DNA extraction method (including CTAB method, SDS method, guanidine salt method, phenol method, column method, etc.) is relatively low in cost, and the extracted product can also meet the requirements of some experiments. The extraction process mainly includes the lysis of cells, extraction with organic solvents (such as chloroform, isoamyl alcohol, etc.), isopropanol or ethanol precipitation, high-speed centrifugation, ethanol washing, elution and other steps. However, toxic and harmful reagents such as chloroform, isoamyl alcohol, and β-mercaptoethanol are used in the extraction process, which seriously endangers the health of the experimenters and the safety of the environment. It also requires multi-step centrifugation operation, which is time-consuming and long, and cannot realize automation and high-throughput extraction. Due to the reasons such as long time-consuming, low efficiency or poor product quality, it can no longer meet the requirements of current biotechnology.

[0004] Compared with the traditional genomic DNA extraction method, the advantages of the magnetic bead method are obvious, such as: ① Simple operation, short time, the entire extraction process mainly includes 4 steps: lysis, binding, washing and elution. ② It can be used with automated instruments to achieve automated high-throughput extraction and save labor costs. ③ The process is safe, and no toxic and harmful reagents such as chloroform, isoamyl alcohol and benzene are used, reducing physical harm to experimenters. ④ Magnetic beads can specifically bind to nucleic acids, which helps to increase DNA yield and purity. However, the lysis and washing steps of some kits of the magnetic bead method require the addition of multiple reagents and multiple washings, which are not easy to operate and cumbersome to operate. Some reagents may precipitate after being placed for a long time, which may cause changes in component concentrations in the long run, resulting in unstable extraction. In addition, there is also the problem that the sample coverage is not wide enough, and the extraction effect is not good for more complex seed samples such as cotton. Therefore, it is necessary to develop a kit for extracting plant genomic DNA by magnetic beads. Summary of the invention

[0005] The first aspect of the present invention aims to provide a binding liquid.

[0006] The second aspect of the present invention aims to provide a reagent combination or a kit.

[0007] The third aspect of the present invention aims to provide a system.

[0008] The fourth aspect of the present invention aims to provide a method for extracting genomic DNA.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a binding solution comprising: 0.1-0.7 M sodium salt and 13-21% (m / v, g / mL) polyethylene glycol (PEG).

[0011] Preferably, the sodium salt comprises at least one of sodium chloride, sodium acetate, sodium sulfate and sodium citrate.

[0012] Preferably, the polyethylene glycol comprises at least one of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000 and PEG20000.

[0013] Preferably, the binding solution comprises: 0.2-0.6 M sodium salt and 14-20% (m / v, g / mL) polyethylene glycol.

[0014] A second aspect of the present invention provides a reagent combination or kit, comprising: a lysate and a binding solution according to the first aspect of the present invention;

[0015] The lysate contains: guanidine salt, sodium salt, and non-ionic surfactant.

[0016] Preferably, the guanidine salt in the lysate comprises at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate and guanidine thiocyanate.

[0017] Preferably, the concentration of the guanidine salt in the lysate is 2.4-3.6M.

[0018] Preferably, the sodium salt in the lysate comprises at least one of sodium chloride, sodium acetate, sodium sulfate and sodium citrate.

[0019] Preferably, the concentration of the sodium salt in the lysate is 0.2-0.8M.

[0020] Preferably, the nonionic surfactant in the lysate comprises at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol.

[0021] Preferably, the concentration of the nonionic surfactant in the lysate is 5-11% (v / v).

[0022] Preferably, the lysate further comprises: polyvinyl pyrrolidone.

[0023] Preferably, the polyvinyl pyrrolidone comprises at least one of polyvinyl pyrrolidone K15, polyvinyl pyrrolidone K30, polyvinyl pyrrolidone K60 and polyvinyl pyrrolidone K90.

[0024] Preferably, the concentration of polyvinyl pyrrolidone in the lysate is 2-6% (m / v).

[0025] Preferably, the lysate further comprises: a chelating agent.

[0026] Preferably, the chelating agent comprises: at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid.

[0027] Preferably, the concentration of the chelating agent in the lysate is 4-41 mM.

[0028] Preferably, the lysate further comprises: a buffer salt.

[0029] Preferably, the buffer salt comprises at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane.

[0030] Preferably, the concentration of the buffer salt in the lysate is 4-26 mM.

[0031] Preferably, the reagent combination or kit further comprises: washing solution I.

[0032] Preferably, the washing solution I comprises: guanidine salt, sodium salt, nonionic surfactant and alcohol.

[0033] Preferably, the guanidine salt in the washing solution I comprises at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate and guanidine thiocyanate.

[0034] Preferably, the concentration of the guanidine salt in the washing solution I is 1.4-3.1M.

[0035] Preferably, the sodium salt in the washing solution I comprises at least one of sodium chloride, sodium acetate, sodium sulfate and sodium citrate.

[0036] Preferably, the concentration of the sodium salt in the washing solution I is 0.9-3.3M.

[0037] Preferably, the nonionic surfactant in the washing solution I comprises at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol.

[0038] Preferably, the concentration of the nonionic surfactant in the washing solution I is 1-8% (v / v).

[0039] Preferably, the alcohol in the washing solution I comprises at least one of ethanol, isopropanol and polyethylene glycol.

[0040] Preferably, the concentration of the alcohol in the washing solution I is 38-72% (v / v).

[0041] Preferably, the reagent combination or kit further comprises: washing solution II.

[0042] Preferably, the washing liquid II is alcohol; further, 75-95% (v / v) alcohol.

[0043] Preferably, the alcohol in the washing solution II comprises at least one of ethanol, isopropanol and polyethylene glycol.

[0044] Preferably, the reagent combination or kit further comprises: an eluent.

[0045] Preferably, the eluent comprises at least one of sterile water, enzyme-free water, NF water, DEPC water, and 1×TE.

[0046] Preferably, the reagent combination or kit further comprises: magnetic beads.

[0047] Preferably, the magnetic beads include: nanomagnetic beads.

[0048] Preferably, the magnetic beads include at least one of hydroxyl magnetic beads, carboxyl magnetic beads, glass magnetic beads, amino magnetic beads, tosyl magnetic beads, and epoxy magnetic beads.

[0049] Preferably, the reagent combination or kit is used for extracting plant genomic DNA using the magnetic bead method.

[0050] Preferably, the plant sample may be its root, stem, leaf, flower, fruit and / or seed.

[0051] Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

[0052] A third aspect of the present invention provides a system comprising: at least one of the binding solution of the first aspect of the present invention, the reagent combination or the kit of the second aspect of the present invention; and

[0053] Nucleic acid extraction instrument.

[0054] The fourth aspect of the present invention provides a method for extracting genomic DNA, comprising the steps of using the binding solution of the first aspect of the present invention, the reagent combination or kit of the second aspect of the present invention and / or the system of the third aspect of the present invention.

[0055] Preferably, the genomic DNA is plant genomic DNA.

[0056] Preferably, the method comprises the following steps:

[0057] a1) mixing the sample with the lysis solution, separating the solid and the liquid, and obtaining a supernatant;

[0058] a2) mixing the supernatant obtained in step a1) with the magnetic beads and the binding solution of the first aspect of the present invention to separate the magnetic beads;

[0059] a3) mixing the magnetic beads obtained in step a2) with washing solution I to separate the magnetic beads;

[0060] a4) mixing the magnetic beads obtained in step a3) with washing solution II to separate the magnetic beads;

[0061] a5) mixing the magnetic beads obtained in step a4) with the elution solution, separating the magnetic beads, and obtaining the genomic DNA.

[0062] Preferably, the lysate, magnetic beads, washing solution I, washing solution II and / or elution solution are the lysate, magnetic beads, washing solution I, washing solution II and / or elution solution in the second aspect of the present invention.

[0063] Preferably, the sample is a plant.

[0064] Preferably, the sample may be a root, stem, leaf, flower, fruit and / or seed of a plant.

[0065] Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

[0066] Preferably, steps a2) to a5) use a nucleic acid extraction instrument.

[0067] The beneficial effects of the present invention are:

[0068] The invention provides a binding liquid, which can be used for magnetic beads to adsorb DNA during the process of extracting genomic DNA by magnetic beads, and can improve the concentration and purity of the extracted product obtained during the process of extracting genomic DNA, thereby having a better extraction effect.

[0069] Furthermore, the present invention provides a reagent combination or a kit, comprising the above-mentioned binding solution and a lysis solution; by adding a nonionic surfactant to the lysis solution, while promoting lysis, it is also beneficial to the precipitation of residues after centrifugation, which can improve the situation of floating plant residues on the liquid surface after centrifugation and effectively avoid the negative impact caused by poor supernatant sampling operation; by adding polyvinyl pyrrolidone to the lysis solution, it can not only effectively reduce the pollution of polyphenols by forming an insoluble complex with phenol, but also can combine with polysaccharides to effectively remove polysaccharides; the reagent combination or kit also comprises a washing solution I and a washing solution II, and the washing solution I can remove impurities such as proteins and lipids, and the washing solution II can wash away impurities such as organic salts, that is, the washing solution can remove some of the sugars, phenols, proteins, lipids, organic salts, etc. adsorbed thereon; the reagent combination or kit also comprises an elution solution for eluting DNA;

[0070] The reagent combination or test kit does not contain toxic and harmful reagents such as chloroform, isoamyl alcohol, and β-mercaptoethanol, and the health hazards to experimenters can be minimized; the amount of each component added is moderate, and all can be stored at room temperature (2-30°C), and will not be left for too long or precipitate at low temperatures; the non-specific adsorption characteristics of magnetic beads can be fully utilized to extract high-yield, high-purity, and intact genomic DNA (common plants and / or polysaccharide and polyphenol plants), which fully meets downstream applications such as PCR, enzyme digestion, molecular hybridization, library construction, and Southern hybridization; the extraction reagents of the reagent combination or test kit have a wide coverage and can be applied to common plants and polysaccharide and polyphenol plants; in addition, for soybean samples with high oil content, the extraction results are also better than those of the tested competing products; at the same time, the lysis solution, binding solution and washing solution I in the test kit have good storage stability, and no precipitation occurs after seven months of storage.

[0071] Furthermore, the present invention provides a system comprising: the above-mentioned binding solution, at least one of the above-mentioned reagent combination or kit; and a nucleic acid extractor; which can realize high-throughput automated extraction, and can directly perform automated extraction after sample pre-treatment, and can obtain products without manual operation after being put on the machine, and can also increase the yield of the extracted product without affecting the purity and fragment integrity of the extracted product. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 It is a result diagram of detecting the integrity of the fragments of the extraction products of Example 4 and Comparative Examples 4 to 6 by using agarose gel electrophoresis.

[0073] Figure 2 It is a result figure which detected the fragment integrity of the extraction products of Examples 4 to 6 by agarose gel electrophoresis.

[0074] Figure 3 It is a result diagram of detecting the integrity of the fragments of the extraction products of Examples 4, 7 to 14, and Comparative Examples 7 to 18 using agarose gel electrophoresis.

[0075] Figure 4 It is a result diagram of detecting the integrity of the fragments of the extraction products of Examples 4 and 15 by agarose gel electrophoresis.

[0076] Figure 5 This is the fragment distribution diagram of product 2100 obtained by constructing a library of the extraction products of Examples 4, 7, 11, 12, and 14: wherein 1 is Example 11 (corn seeds); 2 is Example 4 (tomato leaves); 7 is Example 12 (soybean seeds); 8 is Example 14 (cotton seeds); and 9 is Example 7 (pine needles).

[0077] Figure 6 It is a result diagram of detecting the fragment integrity of the extraction products of Example 4 and Comparative Example 20 by using agarose gel electrophoresis.

[0078] Figure 7 This is a graph showing the stability results of the lysate, binding solution, and washing solution I in Example 1 after being placed at room temperature (15-25° C.) for seven months: wherein 1, 2, and 3 are the lysate, binding solution, and washing solution I, respectively. DETAILED DESCRIPTION

[0079] The first aspect of the present invention provides a binding solution comprising: 0.1-0.7 M sodium salt and 13-21% (m / v, g / mL) polyethylene glycol (PEG).

[0080] Preferably, the sodium salt comprises: at least one of sodium chloride, sodium acetate, sodium sulfate, and sodium citrate; further comprises: at least one of sodium chloride, sodium acetate, and sodium citrate; and further comprises sodium chloride.

[0081] Preferably, the polyethylene glycol comprises: at least one of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000, and PEG20000; further PEG8000.

[0082] Preferably, the binding solution comprises: 0.2-0.6 M sodium salt and 14-20% (m / v, g / mL) polyethylene glycol; further comprises: 0.3-0.5 M sodium salt and 15-17% (m / v, g / mL) polyethylene glycol; further comprises: 0.4 M sodium salt and 16% (m / v, g / mL) polyethylene glycol.

[0083] Preferably, the binding solution is used for magnetic beads to adsorb DNA during the process of extracting genomic DNA using the magnetic bead method.

[0084] Preferably, the genomic DNA is plant genomic DNA.

[0085] Preferably, the plant can be a common plant (ie, non-polysaccharide-polyphenol plant, such as corn, soybean, rice, wheat) or a polysaccharide-polyphenol plant (such as strawberry, cotton, rapeseed, pine needles).

[0086] Preferably, the plant sample may be its root, stem, leaf, flower, fruit and / or seed; further may be leaf and / or seed.

[0087] Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

[0088] A second aspect of the present invention provides a reagent combination or kit for extracting plant genomic DNA using a magnetic bead method, comprising: a lysate and a binding solution according to the first aspect of the present invention;

[0089] The lysate contains: guanidine salt, sodium salt, and non-ionic surfactant.

[0090] Preferably, the guanidine salt in the lysate comprises: at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate, and guanidine thiocyanate; further comprises: at least one of guanidine hydrochloride and guanidine isothiocyanate; further comprises: guanidine isothiocyanate.

[0091] Preferably, the concentration of the guanidine salt in the lysate is 2.4-3.6M; further 2.5-3.5M; further 2.9-3.1M; further 3M.

[0092] Preferably, the sodium salt in the lysate comprises: at least one of sodium chloride, sodium acetate, sodium sulfate, and sodium citrate; further comprises: at least one of sodium chloride, sodium acetate, and sodium citrate; and further comprises sodium chloride.

[0093] Preferably, the concentration of the sodium salt in the lysate is 0.2-0.8M; further 0.25-0.75M; further 0.4-0.6M; further 0.5M.

[0094] Preferably, the nonionic surfactant in the lysate comprises: at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol; further comprises: at least one of Triton 100, Tween 20, and ethylphenyl polyethylene glycol; further Tween 20; the nonionic surfactant promotes lysis and is also beneficial to the precipitation of residues after centrifugation.

[0095] Preferably, the concentration of the nonionic surfactant in the lysate is 5-11% (v / v); further 6-10% (v / v); further 7-9% (v / v); further 8% (v / v).

[0096] Preferably, the lysis solution further comprises: polyvinyl pyrrolidone; polyvinyl pyrrolidone can effectively reduce the contamination of phenol by forming an insoluble complex with phenol, and it can also combine with polysaccharides to effectively remove polysaccharides.

[0097] Preferably, the polyvinyl pyrrolidone comprises: at least one of polyvinyl pyrrolidone K15, polyvinyl pyrrolidone K30 (polyvinyl pyrrolidone 40), polyvinyl pyrrolidone K60, and polyvinyl pyrrolidone K90; further polyvinyl pyrrolidone K30.

[0098] Preferably, the concentration of polyvinyl pyrrolidone in the lysate is 2-6% (m / v, g / mL); further 3-5% (m / v, g / mL); further 3.5-4.5% (m / v, g / mL); further 4% (m / v, g / mL).

[0099] Preferably, the lysate further comprises: a chelating agent.

[0100] Preferably, the chelating agent comprises: at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid; further comprises: at least one of ethylenediaminetetraacetic acid and disodium ethylenediaminetetraacetate; further comprises disodium ethylenediaminetetraacetate.

[0101] Preferably, the concentration of the chelating agent in the lysate is 4-41 mM; further 5-40 mM; further 19-21 mM; further 20 mM.

[0102] Preferably, the pH of the chelating agent is 7-9; further 7.5-8.5; further 8.

[0103] Preferably, the lysate further comprises: a buffer salt.

[0104] Preferably, the buffer salt comprises: at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane; further tris(hydroxymethyl)aminomethane.

[0105] Preferably, the concentration of the buffer salt in the lysate is 4-26 mM; further 5-25 mM; further 19-21 mM; further 20 mM.

[0106] Preferably, the pH of the buffer salt is 7-9; further 7.5-8.5; further 8.

[0107] Preferably, the reagent combination or kit further comprises: washing solution I.

[0108] Preferably, the washing solution I comprises: guanidine salt, sodium salt, non-ionic surfactant and alcohol, and impurities such as protein and lipid can be removed by the washing solution I.

[0109] Preferably, the guanidine salt in the washing solution I comprises: at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate, and guanidine thiocyanate; further comprises: at least one of guanidine hydrochloride and guanidine isothiocyanate; further comprises: guanidine hydrochloride.

[0110] Preferably, the concentration of the guanidine salt in the washing solution I is 1.4-3.1 M; further 1.5-3 M; further 1.9-2.1 M; further 2 M.

[0111] Preferably, the sodium salt in the washing solution I comprises: at least one of sodium chloride, sodium acetate, sodium sulfate, and sodium citrate; further comprises: at least one of sodium chloride, sodium acetate, and sodium citrate; and further comprises sodium chloride.

[0112] Preferably, the concentration of the sodium salt in the washing solution I is 0.9-3.3M; further 1-3.2M; further 1.9-2.1M; further 2M.

[0113] Preferably, the nonionic surfactant in the washing solution I comprises: at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol; further comprises: at least one of Triton 100, Tween 20, ethylphenyl polyethylene glycol; and further Tween 20.

[0114] Preferably, the concentration of the nonionic surfactant in the washing solution I is 1-8% (v / v); further 2-7% (v / v); further 3-5% (v / v); further 4% (v / v).

[0115] Preferably, the alcohol in the washing solution I comprises: at least one of ethanol, isopropanol, polyethylene glycol; further ethanol.

[0116] Preferably, the alcohol in the washing liquid I is anhydrous alcohol.

[0117] Preferably, the concentration of the alcohol in the washing solution I is 38-72% (v / v); further 40-70% (v / v); further 49-51% (v / v); further 50% (v / v).

[0118] Preferably, the reagent combination or the kit further comprises: a washing solution II, which can be used to wash away impurities such as organic salts.

[0119] Preferably, the washing liquid II is alcohol; further, 75-95% (v / v) alcohol; further, 75-85% (v / v) alcohol; further, 80% (v / v) alcohol.

[0120] Preferably, the alcohol in the washing solution II comprises: at least one of ethanol, isopropanol, and polyethylene glycol; further ethanol.

[0121] Preferably, the reagent combination or kit further comprises: an eluent.

[0122] Preferably, the eluent comprises: at least one of sterile water, enzyme-free water, NF water, DEPC water, and 1×TE; further 1×TE.

[0123] Preferably, the reagent combination or kit further comprises: magnetic beads.

[0124] Preferably, the magnetic beads include: nanomagnetic beads.

[0125] Preferably, the magnetic beads include: at least one of hydroxyl magnetic beads, carboxyl magnetic beads, glass magnetic beads, amino magnetic beads, tosyl magnetic beads, and epoxy magnetic beads; further include: at least one of hydroxyl magnetic beads and carboxyl magnetic beads; further, they are carboxyl magnetic beads.

[0126] Preferably, the magnetic beads are a magnetic bead suspension; further, a magnetic bead suspension with a solid content of 20 to 30 mg / mL.

[0127] Preferably, the plant can be an ordinary plant (i.e., non-polysaccharide-polyphenol plant, such as corn, soybean, rice, wheat) or a polysaccharide-polyphenol plant (such as strawberry, cotton, rapeseed, pine needle): when the plant is a polysaccharide-polyphenol plant, the lysis solution contains polyvinyl pyrrolidone; when the plant is an ordinary plant (i.e., non-polysaccharide-polyphenol plant), the lysis solution contains or does not contain polyvinyl pyrrolidone.

[0128] Preferably, the plant sample may be its root, stem, leaf, flower, fruit and / or seed; further may be leaf and / or seed.

[0129] Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

[0130] A third aspect of the present invention provides a system comprising: at least one of the binding solution of the first aspect of the present invention, the reagent combination or the kit of the second aspect of the present invention; and

[0131] Nucleic acid extraction instrument.

[0132] Preferably, the nucleic acid extractor is an automated nucleic acid extractor (eg, automated nucleic acid extractor MGISP-NE32 or automated nucleic acid extractor MGISP-NE384).

[0133] Preferably, the system is used for extracting plant genomic DNA using the magnetic bead method.

[0134] Preferably, the plant can be an ordinary plant (i.e., non-polysaccharide-polyphenol plant, such as corn, soybean, rice, wheat) or a polysaccharide-polyphenol plant (such as strawberry, cotton, rapeseed, pine needle): when the plant is a polysaccharide-polyphenol plant, the lysis solution contains polyvinyl pyrrolidone; when the plant is an ordinary plant (i.e., non-polysaccharide-polyphenol plant), the lysis solution contains or does not contain polyvinyl pyrrolidone.

[0135] Preferably, the plant sample may be its root, stem, leaf, flower, fruit and / or seed; further may be leaf and / or seed.

[0136] Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

[0137] The fourth aspect of the present invention provides the use of the binding solution of the first aspect of the present invention, the reagent combination or kit of the second aspect of the present invention and / or the system of the third aspect of the present invention in extracting plant genomic DNA.

[0138] Preferably, the plant can be an ordinary plant (i.e., non-polysaccharide-polyphenol plant, such as corn, soybean, rice, wheat) or a polysaccharide-polyphenol plant (such as strawberry, cotton, rapeseed, pine needle): when the plant is a polysaccharide-polyphenol plant, the lysis solution contains polyvinyl pyrrolidone; when the plant is an ordinary plant (i.e., non-polysaccharide-polyphenol plant), the lysis solution contains or does not contain polyvinyl pyrrolidone.

[0139] Preferably, the plant sample may be its root, stem, leaf, flower, fruit and / or seed; further may be leaf and / or seed.

[0140] Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

[0141] The fifth aspect of the present invention provides a method for extracting genomic DNA, comprising the steps of using the binding solution of the first aspect of the present invention, the reagent combination or kit of the second aspect of the present invention and / or the system of the third aspect of the present invention.

[0142] Preferably, the genomic DNA is plant genomic DNA.

[0143] Preferably, the method comprises the following steps:

[0144] a1) mixing the sample with the lysis solution, separating the solid and the liquid, and obtaining a supernatant;

[0145] a2) mixing the supernatant obtained in step a1) with the magnetic beads and the binding solution of the first aspect of the present invention to separate the magnetic beads;

[0146] a3) mixing the magnetic beads obtained in step a2) with washing solution I to separate the magnetic beads;

[0147] a4) mixing the magnetic beads obtained in step a3) with washing solution II to separate the magnetic beads;

[0148] a5) mixing the magnetic beads obtained in step a4) with the elution solution, separating the magnetic beads, and obtaining the genomic DNA.

[0149] Preferably, the lysate, magnetic beads, washing solution I, washing solution II and / or elution solution are the lysate, magnetic beads, washing solution I, washing solution II and / or elution solution in the second aspect of the present invention.

[0150] Preferably, the sample is a plant.

[0151] Preferably, the plant can be an ordinary plant (i.e., non-polysaccharide-polyphenol plant, such as corn, soybean, rice, wheat) or a polysaccharide-polyphenol plant (such as strawberry, cotton, rapeseed, pine needle): when the plant is a polysaccharide-polyphenol plant, the lysis solution contains polyvinyl pyrrolidone; when the plant is an ordinary plant (i.e., non-polysaccharide-polyphenol plant), the lysis solution contains or does not contain polyvinyl pyrrolidone.

[0152] Preferably, the plant sample may be its root, stem, leaf, flower, fruit and / or seed; further may be leaf and / or seed.

[0153] Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

[0154] Preferably, steps a2) to a5) use a nucleic acid extraction instrument.

[0155] Preferably, the nucleic acid extractor is the nucleic acid extractor in the third aspect of the present invention.

[0156] Preferably, the mixing in a1) is vortex mixing; further is constant temperature vortex mixing; further is centrifugation at 800-1200 rpm at 50-70°C for 8-12 min, or standing at 50-70°C for 8-12 min and vortexing for 8-12 s every 2-4 min.

[0157] Preferably, the solid-liquid separation method in a1) is centrifugation; further, centrifugation is performed at 11000-15000 rpm for 5-9 min.

[0158] Preferably, the mass volume ratio (mg:μL) of the sample to the lysate is (30-200):2100; further (50-150):2100.

[0159] Preferably, the mixing in a2) is vortex mixing; further, the mixture is allowed to stand at room temperature for 5 to 7 minutes and vortexed for 8 to 12 seconds every 2 to 4 minutes.

[0160] Preferably, the volume ratio of the supernatant, magnetic beads and binding solution in a2) is (25-35):1:(10-20).

[0161] Preferably, the mixing in a3) and a4) is shaking mixing.

[0162] Preferably, the amount of the washing solution I and the washing solution II in step a3) and a4) is 30 to 40 times that of the magnetic beads in step a2).

[0163] Preferably, a4) is repeated 1 to 2 times.

[0164] Preferably, the step of drying is included before a5); further drying is performed for 8 to 12 minutes.

[0165] Preferably, the amount of the eluent in step a5) is 3 to 7 times the amount of the magnetic beads in step a2).

[0166] Preferably, the mixing in a5) is oscillating mixing; further, the mixture is allowed to stand at room temperature for 3 to 7 minutes and vortexed for 8 to 12 seconds every 1 to 2 minutes.

[0167] Preferably, the separation of magnetic beads in a2), a3), a4) and a5) is carried out on a magnetic stand.

[0168] The present invention is further described in detail below through specific examples.

[0169] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0170] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0171] The commercial kit A in this example is named as Magnetic Bead Seed DNA Extraction Kit, purchased from Guangzhou Meiji Biotechnology Co., Ltd., with the item number D6352-02B; the commercial kit B is named as MagMAX TM The plant DNA isolation kit was purchased from Thermo Fisher Scientific with a catalog number of A32549; the commercially available kit C was named the magnetic bead plant genomic DNA extraction kit (DP342) and was purchased from Tiangen Biochemical Technology Co., Ltd. with a catalog number of DP342; the commercially available kit D was named Mag- Plant DNA DS 96Kit M1130 was purchased from OMEGA with the catalog number M1130-00.

[0172] Example 1 A kit for extracting plant DNA using magnetic beads

[0173] A kit for extracting plant group DNA by magnetic bead method, comprising: a lysis solution, a binding solution, a washing solution I, a washing solution II, an elution solution, and a magnetic bead suspension;

[0174] The lysate contains the following components: 3M guanidine thiocyanate, 0.5M sodium chloride, 4% (m / v, g / mL) polyvinyl pyrrolidone 40, 8% (v / v) Tween 20, 20mM tris(hydroxymethyl)aminomethane (pH 8.0), and 20mM ethylenediaminetetraacetic acid (pH 8.0);

[0175] The binding solution contained the following components: 0.4 M sodium chloride and 16% (m / v, g / mL) PEG8000;

[0176] Washing solution I contained the following components: 2 M guanidine hydrochloride, 2 M sodium chloride, 4% (v / v) Tween 20, and 50% (v / v) anhydrous ethanol;

[0177] Washing solution II is 80% (v / v) anhydrous ethanol;

[0178] The eluent was 1×TE;

[0179] The concentration of the magnetic bead suspension is 25 mg / mL (carboxyl magnetic beads TQ01, purchased from MGI, item number 940-000121-00, model 100 mL / bottle).

[0180] Example 2 A kit for extracting plant DNA using magnetic beads

[0181] A kit for extracting plant group DNA by magnetic bead method, comprising: a lysis solution, a binding solution, a washing solution I, a washing solution II, an elution solution, and a magnetic bead suspension;

[0182] The lysate contains the following components: 2.5 M guanidine thiocyanate, 0.25 M sodium chloride, 3% (m / v, g / mL) polyvinyl pyrrolidone 40, 6% (v / v) Tween 20, 5 mM tris (hydroxymethyl) aminomethane (pH 8.0), and 5 mM ethylenediaminetetraacetic acid (pH 8.0);

[0183] The binding solution contained the following components: 0.2 M sodium chloride and 14% (m / v, g / mL) PEG8000;

[0184] Washing solution I contained the following components: 1.5 M guanidine hydrochloride, 1 M sodium chloride, 2% (v / v) Tween 20, and 40% (v / v) anhydrous ethanol;

[0185] Washing solution II is 75% (v / v) anhydrous ethanol;

[0186] The eluent was 1×TE;

[0187] The concentration of the magnetic bead suspension is 25 mg / mL (carboxyl magnetic beads TQ01, purchased from MGI, item number 940-000121-00, model 100 mL / bottle).

[0188] Example 3 A kit for extracting plant DNA using magnetic beads

[0189] A kit for extracting plant group DNA by magnetic bead method, comprising: a lysis solution, a binding solution, a washing solution I, a washing solution II, an elution solution, and a magnetic bead suspension;

[0190] The lysate contains the following components: 3.5 M guanidine thiocyanate, 0.75 M sodium chloride, 5% (m / v, g / mL) polyvinyl pyrrolidone 40, 10% (v / v) Tween 20, 25 mM tris (hydroxymethyl) aminomethane (pH 8.0), and 40 mM ethylenediaminetetraacetic acid (pH 8.0);

[0191] The binding solution contained the following components: 0.6 M sodium chloride and 20% (m / v, g / mL) PEG8000;

[0192] Washing solution I contained the following components: 3 M guanidine hydrochloride, 3.2 M sodium chloride, 7% (v / v) Tween 20, and 70% (v / v) anhydrous ethanol;

[0193] Washing solution II is 95% (v / v) anhydrous ethanol;

[0194] The eluent was 1×TE;

[0195] The concentration of the magnetic bead suspension is 25 mg / mL (carboxyl magnetic beads TQ01, purchased from MGI, item number 940-000121-00, model 100 mL / bottle).

[0196] Example 4 A method for extracting DNA from tomato leaves using magnetic beads

[0197] A method for extracting DNA from tomato leaves using a magnetic bead method, using the kit of Example 1 for extraction, the specific method is as follows:

[0198] (1) Take 50 mg of fresh tomato leaves and place them in a 2 mL centrifuge tube and quickly freeze them in liquid nitrogen for about 10 seconds;

[0199] (2) Place the sample on a grinder with grinding beads and grind into powder. To ensure the consistency of sample input, add PBS (50 μL per portion) according to the required number of portions and mix all the samples evenly.

[0200] (3) Take 50 μL of sample and put it into a centrifuge tube. Add 700 μL of lysis solution into the centrifuge tube, place it in a vortex mixer for oscillation and mix, and place it in a constant temperature mixer at 60°C for 10 minutes at a speed of 1000 rpm.

[0201] (4) Place the centrifuge tube in a centrifuge, set the speed to 13000 rpm, and centrifuge for 7 minutes;

[0202] (5) Pipette 600 μL of supernatant into a new 1.5 mL centrifuge tube;

[0203] (6) Add 20 μL of magnetic bead suspension and 300 μL of binding solution to the centrifuge tube, place the centrifuge tube in a vortex mixer and vortex to mix, let stand at room temperature for 6 minutes, and vortex for 10 seconds every 3 minutes;

[0204] (7) Place the centrifuge tube on the magnetic stand for about 30 seconds. After the magnetic beads are completely absorbed to the tube wall, discard the supernatant and remove the centrifuge tube from the magnetic stand.

[0205] (8) Add 700 μL of washing solution I to the centrifuge tube, vortex to mix, place the centrifuge tube on the magnetic rack for about 30 seconds, wait until the magnetic beads are completely absorbed to the tube wall, discard the supernatant, and remove the centrifuge tube from the magnetic rack;

[0206] (9) Add 700 μL of washing solution II to the centrifuge tube, vortex to mix, place the centrifuge tube on the magnetic rack for about 30 seconds, wait until the magnetic beads are completely absorbed to the tube wall, discard the supernatant, and remove the centrifuge tube from the magnetic rack;

[0207] (10) Repeat step (9) once, open the lid and dry at room temperature for about 10 minutes until there is no liquid left in the tube;

[0208] (11) Add 100 μL of elution buffer TE, vortex to mix, let stand at room temperature for 5 min, and vortex for 10 s every 2 min;

[0209] (12) Place the centrifuge tube on the magnetic rack for about 30 seconds. After the magnetic beads are completely absorbed to the tube wall, transfer the supernatant to a new sterile centrifuge tube to obtain tomato leaf DNA.

[0210] Example 5 A method for extracting DNA from tomato leaves using magnetic beads

[0211] A method for extracting DNA from tomato leaves using a magnetic bead method is the same as that of Example 4, except that the kit of Example 2 is used (the tomato leaf sample in this example is the sample obtained in step (2) of Example 4).

[0212] Example 6 A method for extracting DNA from tomato leaves using magnetic beads

[0213] A method for extracting DNA from tomato leaves using a magnetic bead method is the same as that of Example 4, except that the kit of Example 3 is used (the tomato leaf sample in this example is the sample obtained in step (2) of Example 4).

[0214] Example 7 A method for extracting pine needle DNA using magnetic beads

[0215] A method for extracting pine needle DNA by magnetic beads is the same as that of Example 4, except that steps (1) and (2) are changed to: 50 mg of pine needles are placed in a 2 mL centrifuge tube, and ground into powder on a grinder with grinding beads, and the sample size in step (3) is 50 mg.

[0216] Example 8 A method for extracting DNA from rice leaves using magnetic beads

[0217] A method for extracting DNA from rice leaves using a magnetic bead method is the same as that of Example 4, except that fresh tomato leaves are replaced with rice leaves.

[0218] Example 9 A method for extracting DNA from wheat leaves using magnetic beads

[0219] A method for extracting DNA from wheat leaves using a magnetic bead method is the same as that of Example 4, except that fresh tomato leaves are replaced with wheat leaves.

[0220] Example 10 A method for extracting DNA from rapeseed leaves using magnetic beads

[0221] A method for extracting rapeseed leaf DNA using a magnetic bead method is the same as that of Example 4, except that fresh tomato leaves are replaced with rapeseed leaves.

[0222] Example 11 A method for extracting DNA from corn seeds using magnetic beads

[0223] A method for extracting corn seed DNA using a magnetic bead method is the same as that of Example 4, except that steps (1) and (2) are changed to: placing the corn seeds in a 2 mL centrifuge tube and grinding them into powder on a grinder with grinding beads; and the sample size in step (3) is changed to 30 mg.

[0224] Example 12 A method for extracting soybean seed DNA using magnetic beads

[0225] A method for extracting soybean seed DNA using a magnetic bead method is the same as that of Example 11, except that the corn seeds are replaced by soybean seeds.

[0226] Example 13 A method for extracting DNA from strawberry seeds using magnetic beads

[0227] A method for extracting DNA from strawberry seeds using a magnetic bead method is the same as that in Example 11, except that corn seeds are replaced with strawberry seeds.

[0228] Example 14 A method for extracting cotton seed DNA using magnetic beads

[0229] A method for extracting cotton seed DNA using a magnetic bead method is the same as that of Example 11, except that corn seeds are replaced with cotton seeds.

[0230] Example 15 A method for extracting DNA from tomato leaves using magnetic beads

[0231] A method for extracting DNA from tomato leaves using a magnetic bead method, using MGI's automated nucleic acid extractor MGISP-NE384 and the kit of Example 1 for extraction, the specific method is as follows:

[0232] (1) Before the experiment, the reagents were packaged as shown in Table 1;

[0233] Table 1 Reagent packaging table

[0234]

[0235] (2) 50 mg of fresh tomato leaves were placed in a 2 mL centrifuge tube and quick-frozen in liquid nitrogen for about 10 seconds;

[0236] (3) Place the sample on a grinder with grinding beads and grind it into powder. To ensure that the sample input amount is consistent, add PBS (50 μL per portion) according to the required number of portions and mix all the samples (the tomato leaf sample in this example is the sample obtained in step (2) of Example 4);

[0237] (4) Take 50 μL of sample and put it into a centrifuge tube. Add 700 μL of lysis solution into the centrifuge tube, place it in a vortex mixer for oscillation and mix, and place it in a constant temperature mixer at 60°C for 10 minutes at a speed of 1000 rpm;

[0238] (5) Place the centrifuge tube in a centrifuge, set the speed to 13000 rpm, and centrifuge for 7 minutes; aspirate 600 μL of supernatant to plate position 1, and set the program as shown in Table 2;

[0239] Table 2 Program settings of the automated nucleic acid extractor MGISP-NE384

[0240]

[0241]

[0242] (6) After the extraction is completed, the eluate at position 6 is transferred out, which is the tomato leaf DNA.

[0243] Comparative Example 1 A kit for extracting plant DNA using a modified CTAB method

[0244] A kit for extracting plant group DNA by improved CTAB method, referring to the reagents used in the literature Boiteux LS, Fonseca M, Simon PW. Effects of plant tissue and DNA purification method on randomly amplified polymorphic DNA-based genetic fingerprinting analysis in carrot. Journal of the American Society for Horticultural Science, 1999, 124 (1): 32-38, comprising: CTAB lysis solution, β-mercaptoethanol, chloroform / isoamyl alcohol (v:v=24:1), isopropanol (anhydrous), anhydrous ethanol and 1×TE;

[0245] The CTAB lysis solution contains the following components: 2% (m / v, g / mL) hexadecyltrimethylammonium bromide, 2% (m / v, g / mL) polyvinylpyrrolidone 40, 25 mM tris(hydroxymethyl)aminomethane (pH 8.0), 40 mM ethylenediaminetetraacetic acid (pH 8.0) and 1.4 M sodium chloride.

[0246] Comparative Example 2: A kit for extracting plant DNA using a magnetic bead method

[0247] A kit for extracting plant group DNA by magnetic bead method is the same as Example 1, except that the binding solution in Example 1 is replaced by isopropanol (anhydrous).

[0248] Comparative Example 3: A kit for extracting plant DNA using a magnetic bead method

[0249] A kit for extracting plant group DNA by magnetic bead method is the same as that of Example 1, except that the binding solution in Example 1 is replaced by anhydrous ethanol.

[0250] Comparative Example 4: A method for extracting DNA from tomato leaves using a modified CTAB method

[0251] A method for extracting DNA from tomato leaves using a modified CTAB method, using the kit of comparative example 1 for extraction, the specific method is as follows:

[0252] (1) Take 50 mg of fresh tomato leaves and place them in a 2 mL centrifuge tube and quickly freeze them in liquid nitrogen for about 10 seconds;

[0253] (2) Grind the samples into powder using grinding beads on a grinder. To ensure that the sample input amount is consistent, add PBS (50 μL per portion) according to the required number of portions and mix all the samples evenly (the tomato leaf sample in this comparative example is the sample obtained in step (2) of Example 4);

[0254] (3) Take 50 μL of sample and place it in a centrifuge tube. Add 600 μL of CTAB lysis buffer and 15 μL of β-mercaptoethanol. Mix by shaking and place at 65°C for 30 min.

[0255] (4) Take out the centrifuge tube, let it cool to room temperature, add an equal volume of chloroform / isoamyl alcohol, shake and mix, and centrifuge at 13,000 rpm for 10 min;

[0256] (5) Carefully pipette 500 μL of the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of -20°C precooled anhydrous isopropanol to the centrifuge tube, and let stand at -20°C for 30 min;

[0257] (6) Take out the centrifuge tube, centrifuge at 13000 rpm for 10 min, and discard the supernatant;

[0258] (7) Add 500 μL of anhydrous ethanol, vortex for 30 seconds, centrifuge at 13000 rpm for 5 minutes, and discard the supernatant;

[0259] (8) Repeat step (7) and dry at room temperature for 10 min;

[0260] (9) Add 100 μL of elution buffer (1×TE) and vortex mix for 1 min to obtain tomato leaf DNA.

[0261] Comparative Example 5: A method for extracting DNA from tomato leaves using a magnetic bead method

[0262] A method for extracting DNA from tomato leaves using a magnetic bead method is the same as that of Example 4, except that the kit of Comparative Example 2 is used (the tomato leaf sample in this comparative example is the sample obtained in step (2) of Example 4).

[0263] Comparative Example 6: A method for extracting DNA from tomato leaves using a magnetic bead method

[0264] A method for extracting DNA from tomato leaves using a magnetic bead method is the same as that of Example 4, except that the kit of Comparative Example 3 is used (the tomato leaf sample in this comparative example is the sample obtained in step (2) of Example 4).

[0265] Comparative Example 7: A method for extracting DNA from corn seeds

[0266] A method for extracting DNA from corn seeds, the sample dosage is the same as that in Example 11, commercially available kit A is used, and the method is referred to the instructions (the corn seed sample in this comparative example is the sample obtained in Example 11).

[0267] Comparative Example 8: A method for extracting DNA from corn seeds

[0268] A method for extracting DNA from corn seeds, the sample dosage is the same as that in Example 11, a commercially available kit B is used, and the method is referred to the instructions (the corn seed sample in this comparative example is the sample obtained in Example 11).

[0269] Comparative Example 9: A method for extracting soybean seed DNA

[0270] A method for extracting soybean seed DNA, the sample dosage is the same as that in Example 12, commercially available kit A is used, and the method is referred to the instructions (the soybean seed sample in this comparative example is the sample obtained in Example 12).

[0271] Comparative Example 10 A method for extracting soybean seed DNA

[0272] A method for extracting soybean seed DNA, the sample dosage is the same as that in Example 12, a commercially available kit B is used, and the method is referred to the instructions (the soybean seed sample in this comparative example is the sample obtained in Example 12).

[0273] Comparative Example 11 A method for extracting DNA from strawberry seeds

[0274] A method for extracting DNA from strawberry seeds, the sample dosage is the same as that in Example 13, commercially available kit A is used, and the method is referred to the instructions (the strawberry seed sample in this comparative example is the sample obtained in Example 13).

[0275] Comparative Example 12: A method for extracting DNA from strawberry seeds

[0276] A method for extracting DNA from strawberry seeds, the sample dosage is the same as that in Example 13, a commercially available kit B is used, and the method is referred to the instructions (the strawberry seed sample in this comparative example is the sample obtained in Example 13).

[0277] Comparative Example 13: A method for extracting cotton seed DNA

[0278] A method for extracting cotton seed DNA, the sample dosage is the same as that in Example 14, commercially available kit A is used, and the method is referred to the instructions (the cotton seed sample in this comparative example is the sample obtained in Example 14).

[0279] Comparative Example 14: A method for extracting cotton seed DNA

[0280] A method for extracting cotton seed DNA, the sample dosage is the same as that in Example 14, a commercially available kit B is used, and the method is referred to the instructions (the cotton seed sample in this comparative example is the sample obtained in Example 14).

[0281] Comparative Example 15 A method for extracting pine needle DNA

[0282] A method for extracting DNA from pine needles, the sample dosage is the same as that in Example 7, a commercially available kit C is used, and the method is referred to the instruction manual.

[0283] Comparative Example 16 A method for extracting pine needle DNA

[0284] A method for extracting DNA from pine needles, the sample dosage is the same as in Example 7, a commercially available kit D is used, and the method is referred to the instruction manual.

[0285] Comparative Example 17 A method for extracting DNA from tomato leaves

[0286] A method for extracting DNA from tomato leaves, the sample dosage is the same as that in Example 4, a commercially available kit C is used, and the method is referred to the instructions (the tomato leaf sample in this comparative example is the sample obtained in Example 4).

[0287] Comparative Example 18: A method for extracting DNA from tomato leaves

[0288] A method for extracting DNA from tomato leaves, the sample dosage is the same as that in Example 4, a commercially available kit D is used, and the method is referred to the instructions (the tomato sample in this comparative example is the sample obtained in Example 4).

[0289] Comparative Example 19 A kit for extracting plant DNA using a magnetic bead method

[0290] A kit for extracting plant group DNA by magnetic bead method, which is the same as Example 1, except that the binding solution in Example 1 is replaced by the nucleic acid binding solution in Example 2 of patent document CN109055359A (i.e., a binding solution containing 4M NaCl and 20% (w / v) PEG 8000 (PH=2)).

[0291] Comparative Example 20 A method for extracting DNA from tomato leaves using a magnetic bead method

[0292] A method for extracting DNA from tomato leaves using a magnetic bead method is the same as that of Example 4, except that the kit of Comparative Example 19 is used (the tomato leaf sample in this comparative example is the sample obtained in step (2) of Example 4).

[0293] Effect Example Performance Verification of a Kit for Extracting Plant Genomic DNA Using Magnetic Beads

[0294] 1) Example 4 and Comparative Examples 4 to 6 were used as the same batch for the experiment (i.e., the samples were the same). The concentration and purity of the extracted products of Example 4 and Comparative Examples 4 to 6 were detected by NanoDrop8000 and Qubit, respectively. The fragment integrity of the extracted products of Example 4 (this kit) and Comparative Examples 4 to 6 (Comparative Example 4: improved CTAB method; Comparative Example 5: Comparative Example a; Comparative Example 6: Comparative Example b) was detected by agarose gel electrophoresis. The results are shown in Table 3. Figure 1 As shown in Table 3, it can be seen that the concentration of the product extracted by the kit provided by the present invention (Example 4) is higher than that of Comparative Examples 5 to 6, and is equivalent to Comparative Example 4 (modified CTAB method). It can be seen that the present invention has a better extraction effect by adopting a combination solution composed of sodium salt and PEG, and the purity of the product extracted by the kit provided by the present invention (Example 4) is significantly higher than that of Comparative Examples 4 to 6, and fully meets the requirements of downstream applications such as library construction and qPT-PCR. The concentration detection results of NanoDrop 8000 and Qubit are also the closest, without interference from unnecessary impurities; and the product (genomic DNA) extracted by the kit of the present invention (Example 4) has good integrity, a clear main band, and no obvious tailing phenomenon.

[0295] Table 3 Comparison of test results of the extracted products of Example 4 and Comparative Examples 4 to 6

[0296]

[0297] 2) Experiments were conducted using Examples 4 to 6 as the same batch (i.e., the samples were the same). The concentration and purity of the extracted products of Examples 4 to 6 were detected using NanoDrop 8000, and the fragment integrity of the extracted products of Examples 4 to 6 (Example 4: the kit of the present invention; Example 5: the lowest concentration used; Example 6: the highest concentration used) was detected using agarose gel electrophoresis. The results are shown in Table 4, Figure 2As shown: the effects of Examples 5 to 6 are equivalent to those of Example 4 (there is no significant difference in concentration and purity, and the main bands of the electrophoresis diagram are clear without obvious tailing). The concentration of the extracted product of this batch of Example 4 is significantly different from that in 1) due to the difference in samples (the same is true for other batches below), for example: the difference between old leaves and new leaves will lead to differences in their DNA content.

[0298] Table 4 Comparison of test results of the extracted products of Examples 4 to 6

[0299]

[0300]

[0301] 3) Example 4, Comparative Examples 17 and 18, Example 7, Comparative Examples 15 and 16 (in order to ensure that the sample input is as consistent as possible, the same pine needle is selected as much as possible when weighing), Example 11, Comparative Examples 7 and 8, Example 12, Comparative Examples 9 and 10, Example 13, Comparative Examples 11 and 12, Example 14, Comparative Examples 13 and 14, Example 8, Example 9 and Example 10 are the same batch for experiment (that is, except for the pine needles, the other samples are the same, wherein the samples contain 4 kinds of plant seeds and 5 kinds of leaves, including strawberry seeds, cotton seeds, rape leaves and pine needles containing polysaccharides and polyphenols, and soybean seeds with high oil content), and the concentration and purity of the extracted products of Examples 4, 7 to 14 and Comparative Examples 7 to 18 are respectively detected by NanoDrop8000, and the concentration and purity of the extracted products of Examples 4, 7 to 14 and Comparative Examples 7 to 18 are respectively detected by agar. Glycogel electrophoresis was performed on the extraction products of Examples 4, 7-14, and Comparative Examples 7-18 (Example 4: the kit of the present invention in tomato leaves; Examples 7-14: the kit of the present invention in pine needles, rice leaves, wheat leaves, rape leaves, corn seeds, soybean seeds, strawberry seeds, and cotton seeds, respectively; Comparative Examples 7, 9, 11, and 13: the commercially available kit A in corn seeds, soybean seeds, strawberry seeds, and cotton seeds, respectively; Comparative Examples 8, 10, 12, and 14: the commercially available kit B in corn seeds, soybean seeds, strawberry seeds, and cotton seeds, respectively; Comparative Examples 15 and 17: the commercially available kit C in pine needles and tomato leaves, respectively; Comparative Examples 16 and 18: the commercially available kit D in pine needles and tomato leaves, respectively) to detect the fragment integrity. The results are shown in Table 5. Figure 3As shown: the genomic DNA purity A260 / A280 of pine needles, rape leaves, rice leaves, wheat leaves and cotton seeds extracted by the kit provided by the present invention is all above 1.8, and A260 / A230 is all above 1.6, and the concentration and purity (A260 / A280 and A260 / A230) of the extraction product of the polysaccharide and polyphenol plant-pine needles are significantly better than those of competing products (commercially available kit C and commercially available kit D), and the concentration and purity (A260 / A280 and A260 / A230) of the extraction product of cotton seeds are better than those of commercially available kit B, The concentration and purity (A260 / A230) are better than those of the commercial kit A; although the genomic DNA purity A260 / A280 of corn seeds, soybean seeds, strawberry seeds, and tomato leaves extracted by the kit provided by the present invention is below 1.8 and / or A260 / A230 is below 1.6, except that the purity A260 / A280 of the extraction product of corn seeds extracted by the kit provided by the present invention is slightly lower than that of the commercial kit B, and the concentration of the extraction product of soybean seeds is slightly lower than that of the commercial kit B, the others are better than the competing products (commercial kit A and commercial kit B); and through Figure 3 It can be seen that the gel electrophoresis spotting wells of the extraction product of the kit provided by the present invention are clean, the main band is clear, and there is no obvious tailing phenomenon, while the extraction product of the commercially available kit B is dispersed in the spotting wells and may contain more impurities. In summary, the kit provided by the present invention is superior to the competing products (commercially available kits A, B, C, and D) as a whole.

[0302] Table 5 Comparison of test results of the extracted products of Examples 4, 7, 11 to 14 and Comparative Examples 7 to 18

[0303]

[0304]

[0305]

[0306] 4) Example 4 (portable) and Example 15 (MGISP-NE384) were used as the same batch for the experiment (i.e., the samples were the same). The concentration and purity of the extracted products of Example 4 and 15 were detected by NanoDrop8000, and the fragment integrity of the extracted products of Example 4 and 15 was detected by agarose gel electrophoresis. The results are shown in Table 6. Figure 4 As shown: the purity of the extraction products of Example 4 and Example 15 is equivalent, but the concentration of the extraction product of Example 15 is increased by about 36% compared with Example 4 (that is, the output of the extraction product is increased by about 36%), and the gel electrophoresis spot wells of the extraction product are all clean, the main band is clear, and there is no obvious tailing phenomenon; at the same time, Example 15 can extract 384 samples at the same time, which greatly saves labor costs, so when the extraction amount is large, it can be carried out in conjunction with the instrument.

[0307] Table 6 Comparison of test results of the extracted products of Examples 4 and 15

[0308]

[0309] 5) Select the genomic DNA of Example 4, Comparative Examples 17, 18, Example 14, Comparative Examples 13, 14, Examples 7, 11, 12, and 14 in 3) for library construction and PCR amplification

[0310] a1) Take part of the genomic DNA of Example 4, Comparative Examples 17, 18, Example 14, Comparative Examples 13, 14 in 3) for qRT-PCR (the quantitative kit is selected from Beijing Qingke Biotechnology Co., Ltd. Master qPCR Mix (SYBR Green I with UDG), the method is carried out according to the instructions), and the primer β-Actin (see Table 7) is used for both tomato leaves and cotton seeds. The results are shown in Table 7: the quantification result of tomato leaf genomic DNA of the kit provided by the present invention (Example 4) is at the same level as that of the competing products (Comparative Examples 17 and 18), and the quantification result of cotton seed genomic DNA is equivalent to that of the competing product (commercially available kit B) and is better than that of the competing product (commercially available kit A). Therefore, the genomic DNA extracted by the kit of the present invention can be applied to qRT-PCR without PCR inhibitors.

[0311] Table 7 qRT-PCR primer sequences and control results (average of 3 replicates)

[0312]

[0313] a2) The MGIEasy DNA library preparation reagent kit (Cat. No. 1000006988) manufactured by Wuhan MGI was used to construct libraries for Examples 4, 7, 11, 12, and 14 in 3), and the samples after library construction were sequenced and verified using the MGISEQ 200RS high-throughput sequencing reagent kit FCL PE100 (Cat. No. 1000019844) (the DNA products used for library construction were mixed samples repeated 3 times, and the library construction steps were only 1 (soybean, cotton, pine needle) or 2 repeats (corn and tomato, all samples were operated together), the sequencer was MGISEQ-200, and the experimental method was carried out according to the instructions of the kit. No abnormal phenomenon occurred during the library construction process, and the fragments of the library construction products were distributed in the range of 350 to 400 bp (see Figure 5 ), the library concentration shown in Table 8 fully meets the subsequent cyclization requirements, and the concentration after cyclization is 1.09 ng / μL, which meets the requirements for the machine. The total reads and sequencing quality Q30 after sequencing are both normal. Therefore, the genomic DNA product extracted by the kit provided by the present invention can meet the requirements of library construction and sequencing.

[0314] Table 8 Summary of library construction and sequencing results

[0315]

[0316] 6) Example 4 and Comparative Example 20 were used as the same batch for the experiment (i.e., the samples were the same). The concentration and purity of the extracted products of Example 4 and Comparative Example 20 were detected by NanoDrop8000, and the fragment integrity of the extracted products of Example 4 and Comparative Example 20 was detected by agarose gel electrophoresis. The results are shown in Table 9. Figure 6 As shown in Table 9, it can be seen that the concentration and purity of the product extracted by the kit (Example 4) provided by the present invention are significantly higher than those of Comparative Example 20, and fully meet the requirements of downstream applications such as library construction and qPT-PCR; from the results in 2), it can be seen that the effects of Examples 5 to 6 are equivalent to those of Example 4, therefore, it can be expected that the concentration and purity of the products extracted by Examples 5 to 6 are significantly higher than those of Comparative Example 20; it can be seen that the present invention has a better extraction effect by limiting the concentrations of sodium salt and polyethylene glycol; and the product (genomic DNA) extracted by the kit (Example 4) of the present invention has good integrity, a clear main band, and no obvious tailing phenomenon; at the same time, the lysate, binding solution, and washing solution I of the present invention (Example 4) did not show precipitation at the bottle mouth after being stored at room temperature (15 to 25°C) for seven months ( Figure 7 ), accordingly, no precipitation appeared in the bottle; the concentration of the extracted product of this batch of Example 4 is quite different from that of 1), 2), and 3) due to the difference in samples, for example, the difference between old leaves and new leaves will lead to the difference in their DNA content.

[0317] Table 9 Comparison of test results of the extracted products of Example 4 and Comparative Example 20

[0318]

[0319] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A binding solution comprising: 0.1-0.7 M sodium salt and 13-21% (m / v) polyethylene glycol.

2. The binding liquid according to claim 1, characterized in that: The sodium salt comprises: at least one of sodium chloride, sodium acetate, sodium sulfate and sodium citrate; Preferably, the polyethylene glycol comprises at least one of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000 and PEG20000; Preferably, the binding solution comprises: 0.2-0.6 M sodium salt and 14-20% (m / v) polyethylene glycol.

3. A reagent combination or kit, comprising: a lysate and the binding solution according to any one of claims 1 to 2; The lysate contains: guanidine salt, sodium salt, and non-ionic surfactant.

4. The reagent combination or kit according to claim 3, characterized in that: The guanidine salt in the lysate comprises at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate, and guanidine thiocyanate; Preferably, the concentration of the guanidine salt in the lysate is 2.4 to 3.6 M; Preferably, the sodium salt in the lysate comprises: at least one of sodium chloride, sodium acetate, sodium sulfate, and sodium citrate; Preferably, the concentration of the sodium salt in the lysate is 0.2-0.8 M; Preferably, the nonionic surfactant in the lysate comprises: at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol; Preferably, the concentration of the nonionic surfactant in the lysate is 5 to 11% (v / v); Preferably, the lysate further comprises: polyvinyl pyrrolidone; Preferably, the polyvinyl pyrrolidone comprises: at least one of polyvinyl pyrrolidone K15, polyvinyl pyrrolidone K30, polyvinyl pyrrolidone K60 and polyvinyl pyrrolidone K90; Preferably, the concentration of polyvinyl pyrrolidone in the lysate is 2-6% (m / v); Preferably, the lysate further comprises: a chelating agent; Preferably, the chelating agent comprises: at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid; Preferably, the concentration of the chelating agent in the lysate is 4 to 41 mM; Preferably, the lysate further comprises: a buffer salt; Preferably, the buffer salt comprises: at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane; Preferably, the concentration of the buffer salt in the lysate is 4-26 mM.

5. The reagent combination or kit according to claim 4, characterized in that: The reagent combination or kit further comprises: washing solution I; Preferably, the washing solution I comprises: guanidine salt, sodium salt, nonionic surfactant and alcohol; Preferably, the reagent combination or kit further comprises: washing solution II; Preferably, the washing liquid II is an alcohol; Preferably, the reagent combination or kit further comprises: an eluent; Preferably, the reagent combination or kit further comprises: magnetic beads.

6. The reagent combination or kit according to claim 5, characterized in that: The guanidine salt in the washing solution I comprises at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate, and guanidine thiocyanate; Preferably, the concentration of the guanidine salt in the washing solution I is 1.4 to 3.1 M; Preferably, the sodium salt in the washing solution I comprises: at least one of sodium chloride, sodium acetate, sodium sulfate, and sodium citrate; Preferably, the concentration of the sodium salt in the washing solution I is 0.9 to 3.3 M; Preferably, the nonionic surfactant in the washing solution I comprises: at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol; Preferably, the concentration of the nonionic surfactant in the washing solution I is 1 to 8% (v / v); Preferably, the alcohol in the washing solution I comprises: at least one of ethanol, isopropanol, and polyethylene glycol; Preferably, the concentration of the alcohol in the washing solution I is 38-72% (v / v); Preferably, the alcohol in the washing solution II comprises: at least one of ethanol, isopropanol, and polyethylene glycol; Preferably, the eluent comprises: at least one of sterile water, enzyme-free water, NF water, DEPC water, and 1×TE; Preferably, the magnetic beads include: nanomagnetic beads; Preferably, the magnetic beads include at least one of hydroxyl magnetic beads, carboxyl magnetic beads, glass magnetic beads, amino magnetic beads, tosyl magnetic beads, and epoxy magnetic beads.

7. The reagent combination or kit according to any one of claims 3 to 6, characterized in that: The reagent combination or kit is used for extracting plant genomic DNA using a magnetic bead method; Preferably, the plant sample is its root, stem, leaf, flower, fruit and / or seed; Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

8. A system comprising: at least one of the binding solution according to any one of claims 1 to 2, the reagent combination or the kit according to any one of claims 3 to 7; and Nucleic acid extraction instrument.

9. A method for extracting genomic DNA, comprising the steps of using the binding solution according to any one of claims 1 to 2, the reagent combination or kit according to any one of claims 3 to 7 and / or the system according to claim 8.

10. The method according to claim 9, characterized in that: The method comprises the following steps: a1) mixing the sample with the lysis solution, separating the solid and the liquid, and obtaining a supernatant; a2) mixing the supernatant obtained in step a1) with magnetic beads and the binding solution according to any one of claims 1 to 2, and separating the magnetic beads; a3) mixing the magnetic beads obtained in step a2) with washing solution I to separate the magnetic beads; a4) mixing the magnetic beads obtained in step a3) with washing solution II to separate the magnetic beads; a5) mixing the magnetic beads obtained in step a4) with an eluent, separating the magnetic beads, and obtaining the genomic DNA; Preferably, the lysate, magnetic beads, washing solution I, washing solution II and / or elution solution are the lysate, magnetic beads, washing solution I, washing solution II and / or elution solution in any one of claims 3 to 7; Preferably, steps a2) to a5) use a nucleic acid extraction instrument; Preferably, the sample is a plant; Preferably, the sample is a plant root, stem, leaf, flower, fruit and / or seed; Preferably, the genomic DNA is used for PCR, restriction digestion, molecular hybridization, library construction or Southern hybridization.

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