Kit for extracting plant genome DNA based on magnetic beads and application

Abstract

The invention provides a kit for extracting plant genome DNA based on magnetic beads and application. The kit is composed of a lysis solution, a binding solution, a washing solution I, a washing solution II, an eluent and magnetic beads. The cracking combination solution comprises at least one of lauryl sodium sulfate, cetyltrimethylammonium bromide, guanidine hydrochloride, guanidine isothiocyanate, citric acid, trisodium citrate, Tween 20, polyvinylpyrrolidone, sodium chloride, ethylenediamine tetraacetic acid and Tris-HCl; the cracking combination solution comprises at least one of sodium dodecyl sulfate, cetyltrimethylammonium bromide, guanidine hydrochloride, guanidine isothiocyanate, citric acid, trisodium citrate, Tween 20, polyvinylpyrrolidone, sodium chloride, ethylenediamine tetraacetic acid and Tris-HCl; the binding solution comprises at least one of isopropanol, ethanol, guanidine hydrochloride and polyethylene glycol; the washing liquid I is at least one of guanidine hydrochloride, guanidine isothiocyanate, absolute ethyl alcohol, ethylenediaminetetraacetic acid, sodium chloride and Tris-HCl, the washing liquid II is an ethanol aqueous solution, the eluent is a Tris-HCl and ethylenediaminetetraacetic acid aqueous solution, and the magnetic beads are hydroxyl magnetic beads or carboxyl magnetic beads.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to a kit for extracting plant genomic DNA based on the magnetic bead method and its application. Background Art

[0002] DNA (Deoxyribonucleic acid), also known as deoxyribonucleic acid, is the main carrier of genetic information of most organisms on the earth and determines the basic characteristics of organisms. Plants are an important part of the biosphere, and studying the genetic mechanism of plants is one of the important research contents in biology. Extracting high-quality DNA is the first step in molecular biology research and the basis for ensuring the correctness of research results. There is a rich variety of plant species and tissue types, and the material components of tissues such as roots, stems, leaves, fruits, and seeds of different plants are not the same. For example, the leaf fiber content of crops such as corn and wheat is high, the sugar content of fruit tree leaves such as apples and peaches is high, the sugar and phenolic substance content of fruits such as apples, cherries, and plums is high, and the oil content of seeds such as soybeans, corn, sunflowers, and peanuts is high. These impurities will affect the effect of DNA extraction. Researchers often need to optimize the DNA extraction method according to the characteristics of different species and tissues before they can continue downstream research, which greatly affects the research efficiency. Currently, there are few methods that can be compatible with the DNA extraction of different plant species and tissue types. Most patents only develop and optimize the extraction method for specific sample types. For example, CN109136220A conducts method research on Fagaceae plants, uses silica gel powder to prepare a silica gel adsorption solution, and can effectively purify and obtain high-purity DNA; CN113736774A conducts method research on Phaeanthus ebracteatus, and through pretreatment with a polyacrylamide solution and the addition of an anionic surfactant, the pollution situation of impurities such as polysaccharides is greatly improved; CN108546701A conducts method research on cotton samples.

[0003] Common DNA extraction methods mainly include the phenol-chloroform extraction method, the magnetic bead adsorption method, and the adsorption column method. The reagents of the phenol-chloroform method are toxic to both humans and the environment, and centrifugation is required at the same time, so it is impossible to equip high-throughput automated extraction equipment; due to the affinity problem of the affinity column, the adsorption column method can only adsorb large DNA fragments; the magnetic bead method can avoid using harmful reagents such as phenol-chloroform, and can efficiently adsorb DNA fragments and be compatible with high-throughput automated extraction equipment, and has become a hot research technology for nucleic acid extraction. In order to solve the compatibility problem of different plant and tissue samples, and at the same time take into account personnel safety and extraction efficiency, the present invention provides a kit for extracting multi-plant and multi-tissue genomic DNA based on magnetic beads, which can be widely applied to the extraction of genomic DNA from the roots, stems, leaves, seeds, fruits, etc. of various plants. The extraction process is safe and harmless, the extraction speed is fast, and the effect is stable. Summary of the Invention

[0004] One object of the invention is to provide a magnetic bead-based plant genomic DNA extraction kit, which can be widely used for DNA extraction from various plant leaf, seed, fruit, root and rhizome samples, without the need for harmful chemical reagents, saving time and effort, and having high DNA concentration and purity.

[0005] The kit consists of a lysis solution, a binding solution, wash solution I, wash solution II, an elution solution and a magnetic bead solution. The lysis solution is composed of multiple components including sodium dodecyl sulfate, cetyltrimethylammonium bromide, guanidine hydrochloride, guanidine isothiocyanate, citric acid, trisodium citrate, Tween 20, polyvinylpyrrolidone, sodium chloride, ethylenediaminetetraacetic acid, and Tris-HCl. The binding solution is composed of multiple components including isopropanol, ethanol, guanidine hydrochloride, and polyethylene glycol. Wash solution I is composed of multiple components including guanidine hydrochloride, guanidine isothiocyanate, absolute ethanol, ethylenediaminetetraacetic acid, sodium chloride, and Tris-HCl. Wash solution II is an aqueous ethanol solution. The elution solution is an aqueous solution of Tris-HCl and ethylenediaminetetraacetic acid. The magnetic beads are hydroxyl magnetic beads or carboxyl magnetic beads.

[0006] Preferably, the lysis solution, the binding solution, wash solution I, wash solution II, the elution solution and the magnetic bead solution are separately packaged.

[0007] Preferably, the lysis solution is one or more of sodium dodecyl sulfate or cetyltrimethylammonium bromide, with a mass percentage content of 1% - 5%; one or more of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; citric acid with a concentration of 50 - 200 mM; trisodium citrate with a concentration of 50 - 200 mM; Tween 20 with a mass percentage content of 0.5% - 5%; polyvinylpyrrolidone with a mass percentage content of 1% - 5%; sodium chloride with a concentration of 50 - 500 mM; ethylenediaminetetraacetic acid with a concentration of 20 - 100 mM; Tris-HCl with a concentration of 50 - 200 mM;

[0008] Preferably, the binding solution is one or more of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; isopropanol with a mass percentage content of 40% - 100%; absolute ethanol with a mass percentage content of 40% - 100%; polyethylene glycol with a mass percentage content of 1% - 10%;

[0009] Preferably, wash solution I is one or more of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; Tris-HCl with a concentration of 50 - 200 mM; sodium chloride with a concentration of 50 - 500 mM; ethylenediaminetetraacetic acid with a concentration of 20 - 100 mM; absolute ethanol with a volume ratio of 40% - 60%;

[0010] Preferably, wash solution II is absolute ethanol with a volume ratio of 50% - 80%;

[0011] Preferably, the eluent is such that the Tris-HCl concentration is 10 - 30 mM; the ethylenediaminetetraacetic acid concentration is 0.1 - 0.5 mM;

[0012] Preferably, the magnetic beads are hydroxyl magnetic beads or carboxyl magnetic beads, with a particle size of 100 - 1000 nm and a density of 25 - 100 mg / mL.

[0013] More preferably, the lysis solution is such that the guanidine hydrochloride concentration is 3 M; the citric acid concentration is 50 mM; the trisodium citrate concentration is 50 mM; the mass percentage of Tween 20 is 0.5%; the mass percentage of polyvinylpyrrolidone is 1%; the sodium chloride concentration is 100 mM; the ethylenediaminetetraacetic acid concentration is 20 mM; the Tris-HCl concentration is 50 mM;

[0014] More preferably, the binding solution is such that the guanidine hydrochloride concentration is 5 M; the volume ratio of isopropanol is 40%; the volume ratio of absolute ethanol is 40%; the mass percentage of polyethylene glycol is 10%;

[0015] More preferably, the washing solution I is such that the guanidine hydrochloride concentration is 3 M; the Tris-HCl concentration is 50 mM; the sodium chloride concentration is 200 mM; the ethylenediaminetetraacetic acid concentration is 50 mM; the volume ratio of absolute ethanol is 60%;

[0016] More preferably, the washing solution has an absolute ethanol concentration volume ratio of 70%;

[0017] More preferably, the eluent is such that the Tris-HCl concentration is 20 mM and the ethylenediaminetetraacetic acid concentration is 0.4 mM;

[0018] More preferably, the magnetic beads are hydroxyl magnetic beads, with a particle size of 800 nm and a density of 50 mg / mL.

[0019] The second object of the present invention is to provide a method for extracting plant genomic DNA based on the above kit, and the specific steps of the method are as follows:

[0020] ① Take an appropriate amount of sample, mechanically crush it into powder with steel beads, take 100 mg of the powder into a 2 mL centrifuge tube, add 800 μL of lysis buffer, and mix well; ② Place the centrifuge tube in an oven, heat-bath at 70 °C for 20 min, then let it stand for 5 min, and centrifuge at 12,000 rpm for 5 min; ③ Transfer 300 μL of the supernatant to a new 2 mL centrifuge tube, add an equal volume of binding buffer and 10 μL of magnetic beads, mix well and let it stand for 5 min, use a magnetic stand to magnetically attract for 1 min, and discard the supernatant; ④ Add 1000 μL of washing buffer I, wash for 2 min, use a magnetic stand to magnetically attract for 1 min, and discard the supernatant; ⑤ Add 1000 μL of washing buffer II, wash for 2 min, use a magnetic stand to magnetically attract for 1 min, discard the supernatant, and air-dry for 2 min; ⑥ Add 200 μL of elution buffer, elute at 56 °C for 5 min, magnetically attract for 1 min, and take the supernatant to a new centrifuge tube to complete the extraction work.

[0021] The third object of the present invention is to provide an application for extracting genomic DNA from plant samples such as tomato, pepper, watermelon, melon, corn, wheat, cotton seeds and leaves, and leaves of tea tree, poplar tree, peach tree, etc. based on the above-mentioned kit and method.

[0022] Reagents used in the above technical solutions such as guanidine hydrochloride, citric acid, trisodium citrate, sodium chloride, absolute ethanol, isopropanol, etc., and magnetic beads can be selected from various imported or domestic models according to needs and product performance.

[0023] The kit of the present application can be manually operated or prepared in the form of an automatic operation of a multi-well plate using techniques well-known in the art to further improve efficiency. Beneficial effects: This kit is widely applicable to the extraction of genomic DNA from simple tissues such as different plant leaves and polysaccharide polyphenol complex tissues such as seeds, effectively solving the problem of synchronous extraction of different types of samples and making the plant DNA extraction work more convenient; this kit does not use organic solvents such as chloroform and phenol, which can greatly reduce the physical harm to experimental personnel; the extraction method of this kit is compatible with high-throughput automatic extraction workstations, greatly increasing the extraction efficiency; this kit has extremely low cost, a simple and controllable preparation method, and the quality of the extracted DNA meets the experimental requirements of downstream PCR and sequencing, etc., which is beneficial for large-scale extraction and use by scientific research institutes and research institutions. Description of the Drawings

[0024] Figure 1 Quality inspection results of genomic DNA extraction by different main components of lysis buffer on agarose gel electrophoresis; M is Marker.

[0025] Figure 2 Quality inspection results of genomic DNA extraction by different auxiliary components of lysis buffer on agarose gel electrophoresis; M is Marker.

[0026] Figure 3Quality inspection results of DNA extracted with different binding solutions by agarose gel electrophoresis; M is the Marker.

[0027] Figure 4 Quality inspection results of DNA extracted by different extraction methods by agarose gel electrophoresis; M is the Marker.

[0028] Figure 5 Quality inspection results of DNA extracted from different plants and tissues by agarose gel electrophoresis; M is the Marker.

[0029] Figure 6 PCR test effect diagrams of different plant DNAs; a) is the PCR effect diagram of tomato, b) is the PCR effect diagram of pepper, c) is the PCR effect diagram of watermelon, and d) is the PCR effect diagram of melon. Detailed implementation manners

[0030] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0031] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] Tris-HCl and EDTA in the following embodiments are purchased from Sangon Biotech (Shanghai) Co., Ltd.; CTAB and NaCl are purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Ethanol is purchased from Beijing Beihua Fine Chemicals Co., Ltd. The magnetic beads are hydroxyl magnetic beads and are purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0033] Example 1: Preparation of a plant genomic DNA extraction kit based on magnetic beads

[0034] A kit for extracting genomic DNA of different plants and tissues using magnetic beads, the components of the kit are composed of a lysis solution, a binding solution, a washing solution I, a washing solution II, an elution solution and magnetic beads. Using tomato seeds and leaves as samples, the components and concentrations of the lysis solution, the binding solution and the washing solution I are respectively tested for their effects.

[0035] (1) Lysis solution test

[0036] The main components and concentrations of the lysis solution are as follows: one or more of sodium dodecyl sulfate or cetyltrimethylammonium bromide, with a mass percentage content of 1%-5%; one or more of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1-5 M; citric acid with a concentration of 50-200 mM; trisodium citrate with a concentration of 50-200 mM; Tween 20 with a mass percentage content of 0.5%-5%; polyvinylpyrrolidone with a mass percentage content of 1%-5%; sodium chloride with a concentration of 50-500 mM; ethylenediaminetetraacetic acid with a concentration of 20-100 mM; Tris-HCl with a concentration of 50-200 mM.

[0037] The other components are as follows: the binding solution is absolute ethanol; the washing solution I is guanidine hydrochloride with a concentration of 1 M, Tris-HCl with a concentration of 50 mM, sodium chloride with a concentration of 50 mM, ethylenediaminetetraacetic acid with a concentration of 20 mM, and the volume ratio of absolute ethanol is 60%; the washing solution II is 70% ethanol; the elution solution is Tris-HCl with a concentration of 20 mM and ethylenediaminetetraacetic acid with a concentration of 0.4 mM; the magnetic beads are hydroxyl magnetic beads with a particle size of 800 nm and a density of 50 mg / mL. Sodium dodecyl sulfate, cetyltrimethylammonium bromide, trisodium citrate, Tween 20, polyvinylpyrrolidone, ethylenediaminetetraacetic acid, ethanol, isopropanol, polyethylene glycol, and Tris-HCl were purchased from Sangon Biotech (Shanghai) Co., Ltd.; guanidine hydrochloride, guanidine isothiocyanate, citric acid, and sodium chloride were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0038] First, a gradient test was conducted on the main components of the following four lysis solutions, and the gradient settings are shown in Table 1. The extraction method is as follows: ① Take an appropriate amount of the sample, mechanically crush it into powder with a steel bead, take 100 mg of the powder into a 2 mL centrifuge tube, and add 800 μL of the lysis solution and mix well; ② Place the centrifuge tube in an oven, heat bath at 70 °C for 30 min, then let it stand for 5 min, and centrifuge at 12,000 rpm for 10 min; ③ Take 300 μL of the supernatant and transfer it to a new 2 mL centrifuge tube, add an equal volume of the binding solution and 10 μL of magnetic beads, mix well and let it stand for 10 min, use a magnetic stand to magnetically attract for 2 min, and discard the supernatant; ④ Add 1000 μL of the washing solution I, wash for 4 min, use a magnetic stand to magnetically attract for 2 min, and discard the supernatant; ⑤ Add 1000 μL of the washing solution II, wash for 4 min, use a magnetic stand to magnetically attract for 2 min, discard the supernatant, and air dry for 5 min; ⑥ Add 200 μL of the elution solution, elute at 56 °C for 10 min, magnetically attract for 2 min, and take the supernatant into a new centrifuge tube to complete the extraction work. The DNA quality inspection method is as follows: The genomic DNA extracted from the above experiment was inspected by agarose gel electrophoresis and Nanodrop 8000 ultraviolet spectrophotometry to detect the quality of the extracted DNA. The agarose gel concentration is 1%, the DNA loading amount of the sample is 100 ng, M is 20 Kb DNA Marker, the voltage is 120 V, and the electrophoresis time is 30 min.

[0039] Table 1. Preparation Table for Gradient Test of Main Components of Lysis Solution

[0040]

[0041] Using tomato seeds and leaves as samples, 3 technical replicates were selected for each sample type to perform extraction tests on the above reagent protocols and conduct DNA quality inspections. The quality inspection results by ultraviolet spectrophotometry are shown in Table 2, and the quality inspection results by agarose gel electrophoresis are shown in Figure 1 . Considering the quality inspection results of both methods, the DNA concentrations and purities of tomato seeds and leaves extracted with SDS as the main component of the lysis solution are relatively low, and the brightness of the electrophoresis bands is also the lowest; the DNA concentration and purity of tomato leaves extracted with CTAB as the main component of the lysis solution are relatively high, and the electrophoresis bands are also relatively bright, but the DNA concentration and purity of the extracted seeds are low, and the electrophoresis bands are also weak; the DNA concentrations and purities of tomato leaves and seeds extracted with guanidine hydrochloride as the main component of the lysis solution are both relatively high, and the electrophoresis bands are the brightest. Among them, the DNA concentration in Treatment 1 is on the low side, the DNA purity in Treatment 3 is on the low side and the result stability is poor, while the DNA concentration and purity in Treatment 2 are both good and the result stability is relatively high; the DNA concentrations of tomato leaves and seeds extracted with guanidinium isothiocyanate as the main component of the lysis solution are relatively high, but the purity is low, and the electrophoresis bands are serrated, indicating that there are more impurities in the DNA. Considering the DNA concentration, purity, integrity and other indicators of tomato leaves and seeds extracted with different main components and their concentration treatments, it is considered that a guanidine hydrochloride concentration of 3M is most suitable for the lysis step of DNA extraction from polysaccharide polyphenol samples such as seeds and ordinary leaf samples.

[0042] Table 2. Quality Inspection Results Table of DNA Extracted with Different Main Components of Lysis Solution by Ultraviolet Spectrophotometry

[0043]

[0044] Note: * represents significant differences among 3 technical replicates of the same sample under the same treatment; ** represents extremely significant differences among 3 technical replicates of the same sample under the same treatment.

[0045] After determining the main components and their concentrations of the lysis solution, gradient tests were conducted on other auxiliary components of the lysis solution. The gradient settings are shown in Table 3. The DNA extraction method and DNA quality control method refer to the description of the main component test method of the lysis solution.

[0046] Table 3. Preparation Table for Gradient Test of Auxiliary Components of Lysis Solution

[0047]

[0048] Using tomato seeds and leaves as samples, 4 technical replicates were selected for each sample type to perform extraction tests on the above reagent protocols and conduct DNA quality inspections. The quality inspection results by ultraviolet spectrophotometry are shown in Table 4, and the quality inspection results by agarose gel electrophoresis are shown inFigure 2 The quality inspection results by ultraviolet spectrophotometry showed that the DNA concentrations extracted by Treatment 3 and Treatment 2 were high, but the result stability was poor. The DNA concentration extracted by Treatment 1 was low, but the result stability was good. The impurities in all three treatments were less. The results of agarose gel electrophoresis showed that the DNA integrity extracted by Treatment 1 was the highest, followed by Treatment 2, with partial sample degradation. The DNA integrity of Treatment 3 was the worst, with obvious degradation. Considering the two quality inspection methods, it is considered that the reagent formula of Treatment 1 is most suitable for the lysis step of DNA extraction from polysaccharide-polyphenol samples such as seeds and ordinary leaf samples.

[0049] Table 4. Quality inspection results of DNA extraction by ultraviolet spectrophotometry with different auxiliary components of lysis buffer

[0050]

[0051] Note: * represents significant differences among 4 technical replicates of the same sample under the same treatment; ** represents extremely significant differences among 4 technical replicates of the same sample under the same treatment.

[0052] (2) Binding solution test

[0053] The main components of the binding solution are as follows: one or more of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; the mass percentage of isopropanol is 40 - 100%; the mass percentage of ethanol is 40 - 100%; the mass percentage of polyethylene glycol is 1 - 10%. Other components are as follows: the lysis buffer is Treatment 1 in Table 3; Wash Buffer I is guanidine hydrochloride at a concentration of 1 M, Tris-HCl at a concentration of 50 mM, sodium chloride at a concentration of 50 mM, ethylenediaminetetraacetic acid at a concentration of 20 mM, and the volume ratio of absolute ethanol is 60%; Wash Buffer II is 70% ethanol; the elution buffer is Tris-HCl at a concentration of 20 mM and ethylenediaminetetraacetic acid at a concentration of 0.4 mM; the magnetic beads are hydroxyl magnetic beads with a particle size of 800 nm and a density of 50 mg / mL. The gradient test setting scheme of the binding solution is shown in Table 5. The DNA extraction method and DNA quality control method refer to the description of the main component test method of the lysis buffer.

[0054] Table 5. Preparation table for the gradient test of the binding solution

[0055]

[0056] Taking tomato seeds and leaves as samples, 4 technical replicates were selected for each sample type to conduct extraction tests on the above reagent scheme and perform DNA quality inspection. The quality inspection results by ultraviolet spectrophotometry are shown in Table 6, and the quality inspection results of agarose gel electrophoresis are shown in Figure 3。The quality inspection results by ultraviolet spectrophotometry showed that there was little difference in the nucleic acid concentration extracted by different binding solutions, but there was a large difference in purity. The DNA extracted by Treatment 3 had the highest purity, and the result repeatability was relatively stable. The 260 / 230 ratio of DNA in other treatments was relatively high, indicating that there might be degradation or RNA residue. The results of agarose gel electrophoresis showed that the integrity of nucleic acids extracted by different binding solutions was good, but there was obvious RNA residue in Treatments 1, 2, 4, and 5, and there was almost no RNA residue in Treatment 3. Considering the two DNA quality control methods, it was considered that the reagent formula of the binding solution in Treatment 3 was most suitable for the precipitation binding step of DNA extraction from polysaccharide polyphenol samples such as seeds and ordinary leaf samples.

[0057] Table 6. Quality inspection results of DNA extracted by different binding solutions by ultraviolet spectrophotometry

[0058]

[0059] Note: * represents significant differences among 4 technical replicates of the same sample and the same treatment; ** represents extremely significant differences among 4 technical replicates of the same sample and the same treatment.

[0060] (3) Wash solution I test

[0061] The main components of Wash solution I are as follows: one or more of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; Tris-HCl with a concentration of 50 - 200 mM; sodium chloride with a concentration of 50 - 500 mM; ethylenediaminetetraacetic acid with a concentration of 20 - 100 mM; anhydrous ethanol with a volume ratio of 40% - 60%. Other components are as follows: the lysis solution is Treatment 1 in Table 3; the binding solution is Treatment 3 in Table 5; the wash solution II is 70% ethanol; the elution solution is Tris-HCl with a concentration of 20 mM and ethylenediaminetetraacetic acid with a concentration of 0.4 mM; the magnetic beads are hydroxyl magnetic beads with a particle size of 800 nm and a density of 50 mg / mL. The gradient test setting scheme of the binding solution is shown in Table 7. The DNA extraction method and DNA quality control method refer to the description of the main component test method of the lysis solution.

[0062] Table 7. Preparation table for the gradient test of Wash solution I

[0063]

[0064] Taking tomato seeds and leaves as samples, the above reagent scheme was used for extraction testing, and DNA quality inspection was carried out. The results of ultraviolet spectrophotometry quality inspection are shown in Table 8. It can be seen from the results of ultraviolet spectrophotometry quality inspection that the DNA concentration of treatment 2 is the highest, and the result stability is also relatively good. Followed by treatment 4, the DNA concentrations of treatments 1 and 3 are relatively low, and the result stability is also relatively poor. The results of agarose gel electrophoresis quality inspection also show that the DNA homogeneity and integrity of treatments 2 and 4 are good, and the main bands are bright. The main band brightnesses of the DNA of treatments 1 and 3 are different, and the homogeneity is poor. Therefore, based on the comprehensive results of the two quality inspections, it is considered that treatment 2 is most suitable for the washing step in the DNA extraction process of polysaccharide polyphenol samples such as seeds and ordinary leaf samples.

[0065] Table 8. Results of ultraviolet spectrophotometry quality inspection of DNA extracted with different washing solutions I

[0066]

[0067] Note: * represents significant differences among 4 technical replicates of the same sample under the same treatment; ** represents extremely significant differences among 4 technical replicates of the same sample under the same treatment.

[0068] It can be seen from the above test results that CTAB is suitable for extracting DNA from leaf samples, but not for extracting DNA from polysaccharide polyphenol samples such as seeds; the extraction effects of SDS on leaves and seeds are relatively the same, but the concentration is not very high; guanidine salts have good effects in the DNA extraction of leaves and seeds. Among them, guanidine hydrochloride has the best effect, and the extracted DNA bands are bright, with high integrity, high concentration, and good sugar and phenol removal effects. Guanidine isothiocyanate ranks second. After testing the binding solution and washing solution, it is found that guanidine salts are helpful for nucleic acid precipitation and impurity removal, and guanidine hydrochloride has the best effect. Therefore, this patent believes that the lysis solution is treatment 1 in Table 3; the binding solution is treatment 3 in Table 5; the washing solution I is treatment 2 in Table 7; the washing solution II is 70% ethanol; the elution solution is Tris-HCl with a concentration of 20 mM and ethylenediaminetetraacetic acid with a concentration of 0.4 mM; the magnetic beads are hydroxyl magnetic beads with a particle size of 800 nm and a density of 50 mg / mL, which can meet the simultaneous extraction of DNA from simple samples such as leaves and polysaccharide polyphenol samples such as seeds, and the extracted DNA has a high concentration, high purity, good integrity, and few phenolic sugar impurities.

[0069] Example 2: Optimization of the extraction method of a magnetic bead-based plant genomic DNA extraction kit

[0070] Using the kit in Example 1: the lysis solution is treatment 1 in Table 3, the binding solution is treatment 3 in Table 5, and the washing solution I is treatment 2 in Table 7; the extraction method of the plant genomic DNA extraction kit in Example 1 was tested and optimized to shorten the extraction cycle as much as possible and improve the extraction efficiency on the premise of ensuring DNA quality. Different extraction methods are set as follows:

[0071] Method 1: ① Take an appropriate amount of sample, mechanically crush it into powder with steel balls, take 100 mg of the powder into a 2 mL centrifuge tube, add 800 μL of lysis buffer, and mix well; ② Place the centrifuge tube in an oven, heat-bath at 70 °C for 45 min, then let it stand for 5 min, and centrifuge at 12,000 rpm for 10 min; ③ Transfer 300 μL of the supernatant to a new 2 mL centrifuge tube, add an equal volume of binding buffer and 10 μL of magnetic beads, mix well and let it stand for 15 min, use a magnetic stand to magnetically attract for 3 min, and discard the supernatant; ④ Add 1000 μL of Wash Buffer I, wash for 4 min, use a magnetic stand to magnetically attract for 3 min, and discard the supernatant; ⑤ Add 1000 μL of Wash Buffer II, wash for 4 min, use a magnetic stand to magnetically attract for 3 min, and discard the supernatant, repeat the operation once, and air-dry for 5 min; ⑥ Add 200 μL of elution buffer, elute at 56 °C for 15 min, magnetically attract for 3 min, take the supernatant to a new centrifuge tube to complete the extraction work.

[0072] Method 2: ① Take an appropriate amount of sample, mechanically crush it into powder with steel balls, take 100 mg of the powder into a 2 mL centrifuge tube, add 800 μL of lysis buffer, and mix well; ② Place the centrifuge tube in an oven, heat-bath at 70 °C for 30 min, then let it stand for 5 min, and centrifuge at 12,000 rpm for 10 min; ③ Transfer 300 μL of the supernatant to a new 2 mL centrifuge tube, add an equal volume of binding buffer and 10 μL of magnetic beads, mix well and let it stand for 10 min, use a magnetic stand to magnetically attract for 2 min, and discard the supernatant; ④ Add 1000 μL of Wash Buffer I, wash for 4 min, use a magnetic stand to magnetically attract for 2 min, and discard the supernatant; ⑤ Add 1000 μL of Wash Buffer II, wash for 4 min, use a magnetic stand to magnetically attract for 2 min, and discard the supernatant, repeat the operation once, and air-dry for 5 min; ⑥ Add 200 μL of elution buffer, elute at 56 °C for 10 min, magnetically attract for 2 min, take the supernatant to a new centrifuge tube to complete the extraction work.

[0073] Method 3: ① Take an appropriate amount of sample, mechanically crush it into powder with steel balls, take 100 mg of the powder into a 2 mL centrifuge tube, add 800 μL of lysis buffer, and mix well; ② Place the centrifuge tube in an oven, heat-bath at 70 °C for 20 min, then let it stand for 5 min, and centrifuge at 12,000 rpm for 5 min; ③ Transfer 300 μL of the supernatant to a new 2 mL centrifuge tube, add an equal volume of binding buffer and 10 μL of magnetic beads, mix well and let it stand for 5 min, use a magnetic stand to magnetically attract for 1 min, and discard the supernatant; ④ Add 1000 μL of Wash Buffer I, wash for 2 min, use a magnetic stand to magnetically attract for 1 min, and discard the supernatant; ⑤ Add 1000 μL of Wash Buffer II, wash for 2 min, use a magnetic stand to magnetically attract for 1 min, and discard the supernatant, air-dry for 2 min; ⑥ Add 200 μL of elution buffer, elute at 56 °C for 5 min, magnetically attract for 1 min, take the supernatant to a new centrifuge tube to complete the extraction work.

[0074] Taking tomato seeds and leaves as samples, the above reagent scheme was used for extraction testing, and DNA quality inspection was carried out. The quality inspection results by ultraviolet spectrophotometry are shown in Table 9, and the quality inspection results by agarose gel electrophoresis are shown in Figure 4 . It can be seen from the quality inspection results by ultraviolet spectrophotometry that the DNA concentrations extracted by Method 1 and Method 2 are slightly higher than that of Method 3, and the results of A260 / 230 and A260 / 280 are basically the same among the three methods; it can be seen from the quality inspection results by agarose gel electrophoresis that the DNA electrophoresis bands of the three methods are all bright, without degradation, and have high integrity. It shows that the DNA quality extracted by the three methods is relatively ideal and can be used for downstream experiments. Therefore, in order to extract DNA with the highest efficiency, Method 3 was selected as the extraction method of this patent.

[0075] Table 9. Quality inspection results of DNA extracted by different extraction methods by ultraviolet spectrophotometry

[0076]

[0077] Example 3: Application of a kit for extracting plant genomic DNA based on magnetic beads

[0078] Using the kit in Example 1: The lysis solution was Treatment 1 in Table 3, the binding solution was Treatment 3 in Table 5, and the washing solution I was Treatment 2 in Table 7; the extraction method was Method 3 in Example 2; the DNA quality inspection method referred to Example 1. Taking tomato, pepper, watermelon, melon, corn, wheat, cotton seeds and leaves, as well as the leaves of tea tree, poplar tree and peach tree as samples, 3 biological replicates and 3 technical replicates were made for each sample type. DNA extraction was carried out according to the above kit formula and extraction method, and DNA quality inspection was carried out. At the same time, the DNA of tomato, pepper, watermelon and melon was selected to test the PCR amplification effect by SNP molecular markers, and 3 technical replicates were made for each DNA. The PCR method was as follows: The genomic DNA extracted from the above experiment was diluted to 20 ng / μL, and 1 μL was taken and added to the qPCR reaction system to amplify the molecular markers corresponding to each crop (see Sequences 1-12). The 5 μL PCR fluorescence quantitative detector reaction system included: 2.5 μL of 2×KASP Mix (Low Rox) from LGC Company, 20 ng of genomic DNA, 0.07 μL of primer mix (preferred primer mix ratio: 12 μL each of forward primers Primer F1 and Primer F2 at 100 pmol·L-1, 30 μL of reverse primer Primer R at 100 pmol·L-1, 46 μL of ddH2O. The same detection purpose can also be achieved by using other reasonable primer mix ratios), and the remaining volume was made up with ddH2O. According to the operation manual of the fluorescence quantitative PCR instrument AB-Q6, the sample table was edited and the running program was executed.

[0079] DNA was extracted by the above-mentioned protocol and subjected to DNA quality inspection. The results of the ultraviolet spectrophotometry quality inspection are shown in Table 10, and the results of the agarose gel electrophoresis quality inspection are shown in Figure 5 , and the results of the PCR detection are shown in Figure 6 . Through quality inspection by ultraviolet spectrophotometry and agarose gel electrophoresis, DNA could be extracted from the seed and leaf samples of 10 plants. The obtained plant genomic DNA bands were clear, without trailing and miscellaneous bands, and had good parallelism, indicating that the extracted DNA had good integrity, no degradation, high DNA product content and good purity. At the same time, SNP molecular marker tests were carried out on the DNA of tomatoes, peppers, watermelons, and melons. The different genotypes of each marker were closely clustered and clearly typed, and all markers could obtain effective amplification typing results, indicating that the genomic DNA quality of simple samples such as leaves and seeds and polysaccharide polyphenol samples of various crops extracted by the reagents and methods of this patent could meet the requirements of PCR detection.

[0080] Table 10. Results of ultraviolet spectrophotometry quality inspection of DNA extracted from seeds and leaves of different crops

[0081]

[0082] Note: * represents significant differences among 4 technical replicates of the same sample under the same treatment; ** represents extremely significant differences among 4 technical replicates of the same sample under the same treatment.

[0083] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A plant genomic DNA extraction kit based on the magnetic bead method, characterized in that, The components of the kit consist of a lysis solution, a binding solution, Wash Solution I, Wash Solution II, an elution solution, and magnetic beads; the lysis-binding solution includes the lysis solution which contains at least one of sodium dodecyl sulfate, cetyltrimethylammonium bromide, guanidine hydrochloride, guanidine isothiocyanate, citric acid, trisodium citrate, Tween 20, polyvinylpyrrolidone, sodium chloride, ethylenediaminetetraacetic acid, and Tris-HCl; the binding solution includes at least one of isopropanol, ethanol, guanidine hydrochloride, and polyethylene glycol; Wash Solution I is at least one of guanidine hydrochloride, guanidine isothiocyanate, absolute ethanol, ethylenediaminetetraacetic acid, sodium chloride, and Tris-HCl, Wash Solution II is an aqueous ethanol solution, the elution solution is an aqueous solution of Tris-HCl and ethylenediaminetetraacetic acid, and the magnetic beads are hydroxyl magnetic beads or carboxyl magnetic beads.

2. The plant genomic DNA extraction kit based on the magnetic bead method according to claim 1, wherein In the lysis-binding solution, it includes: At least one of sodium dodecyl sulfate or cetyltrimethylammonium bromide, with a mass percentage content of 1% - 5%; at least one of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; citric acid with a concentration of 50 - 200 mM; trisodium citrate with a concentration of 50 - 200 mM; Tween 20 with a mass percentage content of 0.5% - 5%; polyvinylpyrrolidone with a mass percentage content of 1% - 5%; sodium chloride with a concentration of 50 - 500 mM; ethylenediaminetetraacetic acid with a concentration of 20 - 100 mM; Tris-HCl with a concentration of 50 - 200 mM.

3. The plant genomic DNA extraction kit based on the magnetic bead method according to claim 2, wherein In the lysis solution, the concentration of guanidine hydrochloride is 3 M; the concentration of citric acid is 50 mM; the concentration of trisodium citrate is 50 mM; the mass percentage content of Tween 20 is 0.5%; the mass percentage content of polyvinylpyrrolidone is 1%; the concentration of sodium chloride is 100 mM; the concentration of ethylenediaminetetraacetic acid is 20 mM; the concentration of Tris-HCl is 50 mM.

4. The plant genomic DNA extraction kit based on the magnetic bead method according to claim 1, wherein The binding solution includes: at least one of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; isopropanol with a mass percentage content of 40% - 100%; absolute ethanol with a mass percentage content of 40% - 100%; polyethylene glycol with a mass percentage content of 1% - 10%.

5. The plant genomic DNA extraction kit based on the magnetic bead method according to claim 4, wherein The binding solution includes: the concentration of guanidine hydrochloride is 5 M; the volume ratio of isopropanol is 40%; the volume ratio of absolute ethanol is 40%; the mass percentage content of polyethylene glycol is 10%.

6. The plant genomic DNA extraction kit based on the magnetic bead method according to claim 1, wherein One or more of guanidine hydrochloride or guanidine isothiocyanate, with a concentration of 1 - 5 M; Tris-HCl with a concentration of 50 - 200 mM; sodium chloride with a concentration of 50 - 500 mM; ethylenediaminetetraacetic acid with a concentration of 20 - 100 mM; the volume ratio of absolute ethanol is 40% - 60%; Wash Solution II is, the volume ratio of absolute ethanol is 50% - 80%; the elution solution is, Tris-HCl with a concentration of 10 - 30 mM; ethylenediaminetetraacetic acid with a concentration of 0.1 - 0.5 mM; the magnetic beads are hydroxyl magnetic beads or carboxyl magnetic beads, with a particle size of 100 - 1000 nm and a density of 25 - 100 mg / mL.

7. The plant genomic DNA extraction kit based on the magnetic bead method according to claim 6, wherein The washing solution I is as follows: the concentration of guanidine hydrochloride is 3 M; the concentration of Tris-HCl is 50 mM; the concentration of sodium chloride is 200 mM; the concentration of ethylenediaminetetraacetic acid is 50 mM; the volume ratio of absolute ethanol is 60%; the washing solution is: the volume ratio of absolute ethanol concentration is 70%; the elution solution is: the concentration of Tris-HCl is 20 mM, and the concentration of ethylenediaminetetraacetic acid is 0.4 mM; the magnetic beads are hydroxyl magnetic beads with a particle size of 800 nm and a density of 50 mg / mL.

8. A method for extracting plant genomic DNA based on the magnetic bead method, characterized in that, Using the kit according to any one of claims 1-7, comprising the following steps: 1) Put fresh plant leaves into a mortar, add liquid nitrogen and grind into powder. 2) Put the ground sample into a centrifuge tube, add the lysis solution, and mix well by shaking. 3) Put the centrifuge tube into an oven for heat bath; after the heat bath ends, let it stand still, and then centrifuge. 4) Take the supernatant and transfer it to a new centrifuge tube, add the binding solution and magnetic beads, mix well, and let it stand at room temperature. 5) After the adsorption ends, place it on a magnetic stand for magnetic absorption, discard the supernatant; add washing solution I for washing. 6) After one washing, place it on a magnetic stand for magnetic absorption, discard the supernatant; add washing solution II for washing. 7) After two washings, place it on a magnetic stand for magnetic absorption, discard the supernatant; add the elution solution for elution, magnetic absorption 3 times, take the supernatant to a new centrifuge tube to complete the extraction work. 8) Use agarose gel electrophoresis to quality-check the extracted DNA.

9. Application of the kit according to any one of claims 1-7 or a method for extracting plant genomic DNA based on magnetic beads in claim 8 in extracting plant sample genomic DNA.

10. The application according to claim 9, wherein The plant samples are tomato, pepper, watermelon, melon, corn, wheat, cotton seeds and leaves, or leaves of tea tree, poplar tree, and peach tree.

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