Magnetic bead and preparation method and application thereof

Abstract

The invention provides a magnetic bead and a preparation method and application thereof. The surface of the magnetic bead provided by the invention is modified with restriction enzyme and hydroxyl. The surface of the magnetic bead provided by the invention is simultaneously modified with restriction enzyme and nucleic acid binding sites, and integration of plasmid enzyme digestion and extraction can be realized when the magnetic bead is applied to a plasmid enzyme digestion-recovery integrated kit.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a magnetic bead, its preparation method, and its application. Background Technology

[0002] Restriction endonucleases are a class of deoxyribonucleases that recognize specific sites and cleave double-stranded DNA at or near those sites. They are commonly used for the cloning and construction of DNA molecules. Traditional molecular cloning requires cumbersome procedures to construct new vectors. Specific procedures are as follows: Figure 1 As shown. Traditional molecular cloning techniques are mainly achieved through enzyme digestion-ligation. After plasmid digestion, agarose gel electrophoresis is required, and the linearized plasmid bands are excised and recovered to obtain the digestion product, which takes approximately 120 minutes. Summary of the Invention

[0003] Current techniques involving plasmid digestion followed by agarose gel electrophoresis are cumbersome and time-consuming. A traditional method for recovering fragments from the digested products involves extraction with phenol / chloroform followed by ethanol precipitation. This method is not only tedious but also results in some fragment loss during recovery, leading to low recovery rates. Furthermore, residual phenol can interfere with downstream experiments. Ultimately, this results in low experimental success rates and inconveniences researchers.

[0004] To address the problems existing in the prior art, this invention provides a magnetic bead, its preparation method, and its application.

[0005] In a first aspect, the present invention provides a magnetic bead whose surface is modified with a restriction endonuclease and hydroxyl groups.

[0006] Preferably, the restriction endonuclease is one or both of BamHI and HindIII.

[0007] Preferably, the restriction endonuclease is BamHI and HindIII.

[0008] Preferably, the enzyme activity ratio of BamHI modified on the surface of magnetic beads to HindIII modified on the surface of magnetic beads is 1:3-3:1, preferably 1:1;

[0009] Preferably, the magnetic beads have a particle size of 500 nm to 1 μm.

[0010] Secondly, the present invention provides a method for preparing the magnetic beads, the method comprising the following steps: activating and modifying a restriction endonuclease using an activator, and then reacting the activated and modified restriction endonuclease with nano-iron oxide in an alkaline environment to obtain magnetic beads with a surface modified with restriction endonuclease and hydroxyl groups.

[0011] Preferably, the activator is one or a combination of two or more of chloropropyltrimethoxysilane and chloropropyltriethoxysilane; more preferably, the activator is chloropropyltriethoxysilane.

[0012] And / or, on a per-ultra-L / U basis, the ratio of the activator to the restriction endonuclease is 1:2-10;

[0013] And / or, the reaction temperature for activation modification is 20-30℃;

[0014] And / or, the reaction time for activation modification is 0.5-2 h.

[0015] Preferably, the activated and modified restriction endonuclease is reacted with nano-ferric oxide in a reaction solution with a pH of 8-9;

[0016] Preferably, the reaction solution is a Tris reaction solution;

[0017] Preferably, the reaction temperature is 0-8℃;

[0018] Preferably, the reaction time is 20-30 hours.

[0019] Preferably, the restriction endonuclease is one or both of BamHI and HindIII; more preferably, the restriction endonuclease is BamHI and HindIII.

[0020] And / or, based on U / mg, the ratio of activated modified restriction endonuclease to magnetic beads is 1-4:1;

[0021] Preferably, the ratio of activated modified BamHI to magnetic beads is 1-3:1, based on U / mg.

[0022] Preferably, the ratio of activated modified HindIII to magnetic beads is 1-3:1, based on U / mg.

[0023] Thirdly, the present invention provides magnetic beads obtained by the preparation method described above.

[0024] Fourthly, the present invention provides a kit containing the magnetic beads described above or the magnetic beads obtained by the preparation method described above.

[0025] Preferably, the kit contains a magnetic bead enzyme digestion system premix, an enzyme digestion product binding solution, a rinsing solution, and an elution solution;

[0026] The magnetic beads are contained in the premixed solution of the magnetic bead enzymatic digestion system.

[0027] Preferably, the magnetic bead enzymatic digestion system premix contains Tris-acetate, sorbitol, and the magnetic beads;

[0028] Preferably, the concentration of Tris-acetate in the premixed solution of the magnetic bead enzymatic digestion system is 15-30 mM, more preferably 20 mM, preferably the pH value of Tris-acetate is 7.5-8.5, and more preferably the pH value of Tris-acetate is 7.9.

[0029] Preferably, the concentration of sorbitol in the premixed solution of the magnetic bead enzymatic digestion system is 60-80 wt%, more preferably 70 wt%.

[0030] Preferably, the concentration of magnetic beads in the premixed solution of the magnetic bead enzymatic digestion system is 0.5-2 wt%, more preferably 1 wt%.

[0031] Preferably, the magnetic bead enzymatic digestion system premix further contains magnesium acetate, potassium acetate, and BSA;

[0032] More preferably, the concentration of magnesium acetate in the premixed solution of the magnetic bead enzymatic digestion system is 5-15 mM, preferably 10 mM;

[0033] More preferably, the concentration of potassium acetate in the premixed solution of the magnetic bead enzymatic digestion system is 40-60 mM, preferably 50 mM;

[0034] More preferably, the concentration of BSA in the premixed solution of the magnetic bead enzymatic digestion system is 5-15 μg / ml, preferably 10 μg / ml;

[0035] And / or, the enzyme digestion product binding solution contains alcohol and salt;

[0036] Preferably, the volume percentage of alcohol in the enzyme digestion product conjugate is 75-85%, more preferably 80%;

[0037] Preferably, the alcohol is isopropanol, and the salt is ammonium acetate;

[0038] More preferably, the enzyme digestion product conjugate contains 1-2M ammonium acetate, preferably 1.5M ammonium acetate;

[0039] Preferably, the pH of the enzyme digestion product conjugate is 5-6, and more preferably 5.5.

[0040] Fifthly, the present invention provides the application of the magnetic beads described herein, or the magnetic beads obtained by the preparation method described herein, or the kit described herein, in plasmid enzymatic digestion and recovery.

[0041] The present invention has the following beneficial effects:

[0042] 1. The magnetic beads provided by this invention are simultaneously modified with restriction endonuclease and nucleic acid binding sites on their surface, and when applied to a plasmid digestion-recovery integrated kit, they can achieve integrated plasmid digestion and extraction.

[0043] 2. This invention solves the problem of cumbersome and time-consuming plasmid enzyme digestion and recovery steps. Using the kit provided by this invention, only three steps are required from enzyme digestion to recovery of the digested product, taking a total of 20 minutes. Compared with traditional plasmid enzyme digestion and recovery, agarose gel electrophoresis is not required for gel extraction and recovery; the digested product is directly recovered using the adsorption capacity of magnetic beads, significantly improving the recovery efficiency by 50%.

[0044] 3. The kit can be used in the same program of a nucleic acid extractor (HR-Z-II) to complete plasmid digestion and digestion product recovery.

[0045] 4. The integrated plasmid digestion and extraction kit includes a magnetic bead digestion system premix, a digestion product binding solution, a washing buffer, and an elution buffer. The magnetic beads in the premix are modified with both restriction endonucleases and hydroxyl groups. The restriction endonucleases can perform plasmid digestion, and the hydroxyl sites can bind to the digestion products after digestion.

[0046] 5. The premixed solution of the magnetic bead enzymatic digestion system contains magnetic beads capable of enzymatic extraction provided by this invention and a sorbitol solution. The sorbitol solution is hygroscopic, preventing crystallization of the premixed solution at low temperatures. The surface structure of the magnetic beads and the coupled restriction endonuclease are not damaged, and the enzymatic activity of the magnetic beads can be maintained for a long time under low-temperature conditions. Attached Figure Description

[0047] Figure 1 The diagram shows the flowchart of molecular cloning procedures in the prior art.

[0048] Figure 2 The diagram shows the specific operation flowchart for plasmid digestion and fragment recovery using the kit provided by this invention.

[0049] Figure 3 The image shown is a transmission electron microscope (TEM) image of magnetic beads modified with restriction endonucleases BamHI and HindIII according to Example 1.

[0050] Figure 4 The image shown is a transmission electron micrograph of the nano-iron oxide used in Example 1;

[0051] Figure 5 The images shown are infrared spectra of nano-iron oxide and magnetic beads with surface-modified restriction endonucleases and hydroxyl groups in Example 1. In the image, A is the infrared spectrum of nano-iron oxide and B is the infrared spectrum of magnetic beads with surface-modified restriction endonucleases and hydroxyl groups.

[0052] Figure 6 The images shown are the recombinant plasmid maps from Examples 1-3.

[0053] Figure 7The image shows electrophoresis diagrams of recombinant plasmid digested with magnetic beads in Examples 1-3. Lane 1 is the product of recombinant plasmid digested with magnetic beads in Example 2; lane 2 is the product of recombinant plasmid digested with magnetic beads in Example 3; lane 3 is the product of recombinant plasmid digested with magnetic beads in Example 1; and lane 4 is the undigested recombinant plasmid.

[0054] Figure 8 The images show the infrared spectra of nano-iron oxide and magnetic beads with surface-modified restriction endonucleases and hydroxyl groups in Examples 2 and 3; where A is the infrared spectrum of nano-iron oxide, B is the infrared spectrum of magnetic beads with surface-modified restriction endonucleases and hydroxyl groups in Example 2, and C is the infrared spectrum of magnetic beads with surface-modified restriction endonucleases and hydroxyl groups in Example 3.

[0055] Figure 9 The image shown is a spectrum of plasmid PET28a from the example.

[0056] Figure 10 The image shown is an agarose gel electrophoresis diagram of the enzyme digestion product recovery in Example 4. Lane 1 is the enzyme digestion product that was not purified and recovered; lane 2 is the product obtained in Example 4; lane 3 is the enzyme digestion product after normal gel excision and recovery (control); and lane 4 is the PET28a vector fragment that was not digested. Detailed Implementation

[0057] In a first aspect, the present invention provides a magnetic bead whose surface is modified with a restriction endonuclease and hydroxyl groups.

[0058] In one embodiment of the present invention, the restriction endonuclease is one or both of BamHI and HindIII.

[0059] In one specific embodiment of the present invention, the surface of the magnetic beads is modified with restriction endonucleases BamHI and HindIII, as well as hydroxyl groups.

[0060] In this invention, the enzyme activity ratio of BamHI modified on the surface of magnetic beads and HindIII modified on the surface of magnetic beads can be 1:3-3:1, for example, 1:3, 1:2, 1:1, 2:1, 3:1 or a range thereof.

[0061] In one specific embodiment of the present invention, the particle size of the magnetic beads is 500nm-1μm, for example, 500nm, 600nm, 700nm, 800nm, 900nm, 1μm or a range thereof.

[0062] Secondly, the present invention provides a method for preparing the magnetic beads, the method comprising the following steps: activating and modifying a restriction endonuclease using an activator, and then reacting the activated and modified restriction endonuclease with nano-iron oxide in an alkaline environment to obtain magnetic beads with a surface modified with restriction endonuclease and hydroxyl groups.

[0063] In one embodiment of the present invention, the activator is one or a combination of two or more of chloropropyltrimethoxysilane and chloropropyltriethoxysilane. Preferably, chloropropyltriethoxysilane is used as the activator to activate the restriction endonuclease.

[0064] In a specific embodiment of the present invention, the chloropropyl group in the activator chloropropyltriethoxysilane and the amino group on the restriction endonuclease can undergo a rapid substitution reaction, and the triethoxysilane is completely hydrolyzed and coated on the surface of the nano-iron oxide particles. The excess chloropropyl group is hydrolyzed into hydroxyl groups under weakly alkaline conditions.

[0065] In one embodiment of the present invention, the method for preparing magnetic beads provided by the present invention includes the following steps:

[0066] Preparation of activated and modified restriction endonuclease BamHI: BamHI is added to the activator chloropropyltriethoxysilane. The chloropropyl group and the amino group on the restriction endonuclease can undergo a rapid substitution reaction. The mixture is then used for the reaction.

[0067] Preparation of activated and modified restriction endonuclease HindIII: HindIII was added to chloropropyltriethoxysilane and the mixture was reacted.

[0068] The modified solutions of the two restriction endonucleases were added to an alkaline reaction solution, followed by the addition of nano-iron oxide particles. The reaction allowed triethoxysilane to be completely hydrolyzed and coated onto the surface of the nano-iron oxide particles. Excess chloropropyl groups were hydrolyzed into hydroxyl groups under weakly alkaline conditions, resulting in magnetic beads with surface-modified restriction endonucleases and hydroxyl groups provided by this invention.

[0069] In one embodiment of the present invention, the alkaline reaction solution is a Tris reaction solution.

[0070] In one embodiment of the present invention, the alkaline reaction solution is a Tris reaction solution with a pH value of 8-9. For example, the alkaline reaction solution is a Tris reaction solution with a pH value of 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9 or a range thereof.

[0071] In one embodiment of the present invention, the modified solutions of two restriction endonucleases are reacted with nano-iron oxide particles in an alkaline reaction solution at 0-8°C for 20-30 h. For example, the reaction is carried out at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or a range thereof. For example, the reaction time is 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, or a range thereof.

[0072] In one embodiment of the present invention, the obtained surface-modified restriction endonuclease and hydroxyl magnetic beads are magnetically removed from the reaction solution using a magnetic rack, washed twice with pure water, and finally stored in the enzyme digestion system premix solution.

[0073] In one embodiment of the present invention, a kit is provided, the kit containing the magnetic beads described above or the magnetic beads obtained by the preparation method described above.

[0074] The kit provided by this invention can achieve integrated plasmid digestion and recovery.

[0075] In one embodiment of the present invention, the kit provided by the present invention contains a magnetic bead enzyme digestion system premix, an enzyme digestion product binding solution, a rinsing solution, and an elution solution; wherein the magnetic bead enzyme digestion system premix contains magnetic beads provided by the present invention.

[0076] In one embodiment of the present invention, the magnetic bead enzymatic digestion system premix contains Tris-acetate, sorbitol and the magnetic beads;

[0077] In one embodiment of the present invention, the concentration of Tris-acetate in the premixed solution of the magnetic bead enzymatic digestion system is 15-30 mM, for example, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM or a range thereof, preferably 20 mM.

[0078] In one embodiment of the present invention, the pH value of Tris-acetate is 7.5-8.5, for example, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or a range thereof, preferably, the pH value of Tris-acetate is 7.9;

[0079] In one embodiment of the present invention, the concentration of sorbitol in the premixed solution of the magnetic bead enzymatic digestion system is 60-80 wt%, for example, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, or a range thereof.

[0080] In one specific embodiment of the present invention, the concentration of sorbitol in the premixed solution of the magnetic bead enzymatic digestion system is 70 wt%.

[0081] In one embodiment of the present invention, the concentration of magnetic beads in the premixed solution of the magnetic bead enzymatic digestion system is 0.5-2 wt%, for example, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, or a range thereof.

[0082] In one specific embodiment of the present invention, the concentration of magnetic beads in the premixed solution of the magnetic bead enzymatic digestion system is 1 wt%.

[0083] In one specific embodiment of the present invention, the magnetic bead enzymatic digestion system premix further contains magnesium acetate, potassium acetate and BSA;

[0084] In one specific embodiment of the present invention, the concentration of magnesium acetate in the premixed solution of the magnetic bead enzymatic digestion system is 5-15 mM, for example, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM or a range thereof, preferably 10 mM.

[0085] In one specific embodiment of the present invention, the concentration of potassium acetate in the premixed solution of the magnetic bead enzymatic digestion system is 40-60 mM, for example, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM or a range thereof, preferably 50 mM;

[0086] In one specific embodiment of the present invention, the concentration of BSA in the premixed solution of the magnetic bead enzymatic digestion system is 5-15 μg / ml, for example, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml or a range thereof, preferably 10 μg / ml.

[0087] In one embodiment of the present invention, the enzyme digestion product conjugate contains alcohol and salt.

[0088] In one embodiment of the present invention, the volume percentage of alcohol in the enzyme digestion product conjugate is 75-85%.

[0089] In one embodiment of the present invention, the volume percentage of alcohol in the enzyme digestion product conjugate is 80%.

[0090] In one embodiment of the present invention, the alcohol in the enzyme digestion product binding solution is isopropanol.

[0091] In one embodiment of the present invention, the salt in the enzyme digestion product binding solution is ammonium acetate.

[0092] Isopropanol has the effect of precipitating nucleic acids, so the linearized enzyme digestion products can aggregate and form a precipitate under the action of isopropanol, and then be reused.

[0093] In one embodiment of the present invention, the volume fraction of isopropanol in the enzyme digestion product conjugate is 75-85%, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or a range thereof.

[0094] In one specific embodiment of the present invention, the volume fraction of isopropanol in the enzyme digestion product conjugate is 80%.

[0095] In one embodiment of the present invention, the enzyme digestion product conjugate contains 1-2M ammonium acetate, for example, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M or a range thereof.

[0096] In one specific embodiment of the present invention, the enzyme digestion product conjugate contains 1.5M ammonium acetate.

[0097] In one embodiment of the present invention, the pH value of the enzyme digestion product binding solution is 5-6, for example, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 or a range thereof.

[0098] In one specific embodiment of the present invention, the pH value of the enzyme digestion product binding solution is 5.5.

[0099] In one specific embodiment of the present invention, the rinsing solution in the kit contains an alcohol solution, preferably an ethanol solution, preferably 75-85% ethanol by volume, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% or a range thereof.

[0100] In one specific embodiment of the present invention, the elution buffer in the kit contains a TE solution.

[0101] In one embodiment of the present invention, the kit comprises the following components:

[0102] The magnetic bead enzymatic digestion system premix contains 60-80 wt% sorbitol, 5-15 mM magnesium acetate, 40-60 mM potassium acetate, 15-30 mM Tris-acetate, 5-15 μg / ml BSA and 0.5-2 wt% magnetic beads, wherein the pH value of Tris-acetate is 7.5-8.5;

[0103] The enzyme digestion product conjugate contains 75-85% isopropanol and 1-2M ammonium acetate by volume, with a pH of 5-6.

[0104] The rinsing solution contains 75-85% ethanol by volume;

[0105] The eluent contains TE solution.

[0106] In one embodiment of the present invention, the magnetic beads described herein, or the magnetic beads obtained by the preparation method described herein, or the kit described herein, are provided for use in plasmid enzymatic digestion and recovery.

[0107] This invention provides an integrated plasmid digestion and extraction kit, which is of great significance for shortening experimental time, reducing operational steps, and improving fragment recovery rate. The specific operational procedure for plasmid digestion and fragment recovery using the kit provided by this invention is as follows: Figure 2 As shown. Figure 2 As shown, the plasmid digestion and fragment recovery using the kit provided by this invention takes only 20 minutes, which is convenient and quick.

[0108] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the protection scope of the present invention.

[0109] Example 1: A magnetic bead

[0110] Preparation of magnetic beads with surface-modified restriction endonucleases and hydroxyl groups:

[0111] ① Add 20 U BamHI to 10 μL of chloropropyltriethoxysilane and mix and react at room temperature for 1 h to obtain activated BamHI;

[0112] ② Add 20 U HindIII to 10 μL of chloropropyltriethoxysilane and mix and react at room temperature for 1 h to obtain activated HindIII;

[0113] ③ Add the activated BamHI and activated HindIII solutions together to 200uL of Tris reaction solution (pH 8.5), then add 20mg of nano-iron oxide particles (500nm in diameter), and react at 4℃ for 24h.

[0114] ④ After the reaction is complete, use a magnetic rack to remove the reaction solution and wash it twice with 500uL of pure water;

[0115] ⑤ Finally, the modified magnetic beads were stored in 100uL of enzyme digestion system premix.

[0116] Transmission electron microscopy (TEM) image of the magnetic beads prepared in Example 1 is shown below. Figure 3 As shown, the transmission electron microscope image of unmodified nano-Fe3O4 is as follows: Figure 4 As shown, the nano-sized iron oxide particles have a diameter of 500 nm. From... Figure 3 and Figure 4 It can be seen that, compared with nano-iron oxide, the magnetic beads modified with restriction endonucleases exhibit better dispersibility. The infrared spectrum of the magnetic beads prepared in Example 1 after surface modification is shown below. Figure 5 As shown. Figure 5 In the image, A represents the infrared spectrum of nano-ferric oxide. Figure 5 B in the image represents the infrared spectrum of magnetic beads with surface modifications of restriction endonucleases and hydroxyl groups. According to... Figure 5 Compared with the specific absorption peaks in the spectrum, the modified magnetic beads exhibit a different absorption peak at 3420 cm⁻¹. -1 and 3450cm -1 It exhibits absorption peaks at 650 cm⁻¹, including the NH stretching vibration of secondary amines and the OH stretching vibration of nitrogen. -1 and 620cm -1 It exhibits CS absorption peaks and sulfite absorption peaks; in the range of 3300-3500 cm⁻¹ -1 There is a distinct broad peak, mainly due to the interaction between different group vibration modes, primarily caused by a large number of NH and OH stretching vibration peaks. The characteristic absorption peak of NH stretching vibration is caused by the NH group on the restriction endonuclease. The OH stretching vibration peak indicates that the magnetic beads are modified with hydroxyl groups, which are produced by the hydrolysis of chloropropyl groups.

[0117] The obtained magnetic beads were used for enzyme digestion. 1 μg of recombinant plasmid and 5 μL of digestion buffer were added to a PCR tube. The recombinant plasmid pattern is shown below. Figure 6 As shown, the prepared HindIII-BamHI magnetic beads were then added, and sterile water was added to a final volume of 50 μL. The PCR reaction was performed at 37°C for 15 min, followed by inactivation at 80°C for 20 min. After the reaction, 20 μL of the sample was taken and subjected to agarose gel electrophoresis at 240 V for 25 min (1 wt% agarose concentration). The results are shown below. Figure 7 As shown in hole position 3. Figure 7 Well number 4 contains an undigested recombinant plasmid. (For example...) Figure 7 As shown in the diagram for borehole position 3 in the middle section. Figure 6 The recombinant plasmid shown was used as a substrate for enzyme digestion. The digested fragments were 1169bp and 4576bp, respectively. Two bands were shown in the gel image, with no dragging phenomenon and no asterisk activity.

[0118] Example 2: A magnetic bead

[0119] Preparation of magnetic beads with surface-modified restriction endonucleases and hydroxyl groups:

[0120] ① Add 60 U BamHI to 10 μL of chloropropyltriethoxysilane and mix and react at room temperature for 1 h to obtain activated BamHI;

[0121] ② Add 20 U HindIII to 10 μL of chloropropyltriethoxysilane and mix and react at room temperature for 1 h to obtain activated HindIII;

[0122] ③ Add the activated BamHI and activated HindIII solutions together to 200 μL of Tris reaction solution (pH 8.5), then add 20 mg of nano-iron oxide particles (1 μm in diameter), and react at 4 °C for 24 h.

[0123] ④ After the reaction is complete, use a magnetic rack to remove the reaction solution and wash it twice with 500uL of pure water;

[0124] ⑤ Finally, the modified magnetic beads were stored in 100uL of enzyme digestion system premix.

[0125] The obtained magnetic beads were subjected to infrared spectroscopy detection, and the results are as follows: Figure 8 As shown. Figure 8 In the image, A represents the infrared spectrum of nano-ferric oxide. Figure 8 In Example 2, B represents the infrared spectrum of magnetic beads with surface modifications of restriction endonucleases and hydroxyl groups. Figure 8 As shown in B, in the spectrum at 3420 cm⁻¹ -1 and 3450cm -1 It exhibits absorption peaks at 650 cm⁻¹, including the NH stretching vibration of secondary amines and the OH stretching vibration of nitrogen. -1 and 620cm -1 There are CS absorption peaks and sulfite absorption peaks, in the range of 3300-3500 cm⁻¹. -1 There was a distinct broad peak, indicating that the magnetic beads were modified with restriction endonucleases BamHI and HindIII and hydroxyl groups.

[0126] The obtained magnetic beads were used for enzymatic digestion. The substrate and conditions for the enzymatic digestion reaction were the same as in Example 1, and the results are as follows. Figure 7 As shown in the diagram for borehole position 1, since the diagram is as follows... Figure 6 The recombinant plasmid shown was used as a substrate for enzyme digestion. The digested fragments were 1169bp and 4576bp, respectively. Two bands were shown in the gel image, indicating a dragging phenomenon and the presence of asterisk-marked activity.

[0127] Example 3: A magnetic bead

[0128] Preparation of magnetic beads with surface-modified restriction endonucleases and hydroxyl groups:

[0129] ① Add 20 U BamHI to 10 μL of chloropropyltriethoxysilane and mix and react at room temperature for 1 h to obtain activated BamHI;

[0130] ② Add 60 U HindIII to 10 μL of chloropropyltriethoxysilane and mix and react at room temperature for 1 h to obtain activated HindIII;

[0131] ③ Add the activated BamHI and activated HindIII solutions together to 200uL of Tris reaction solution (pH 8.5), then add 20mg of nano-iron oxide particles (500nm in diameter), and react at 4℃ for 24h.

[0132] ④ After the reaction is complete, use a magnetic rack to remove the reaction solution and wash it twice with 500uL of pure water;

[0133] ⑤ Finally, the modified magnetic beads were stored in 100uL of enzyme digestion system premix.

[0134] The obtained magnetic beads were subjected to infrared spectroscopy detection, and the results are as follows: Figure 8 As shown. Figure 8 In the image, A represents the infrared spectrum of nano-ferric oxide. Figure 8 C in the image represents the infrared spectrum of magnetic beads modified with restriction endonucleases and hydroxyl groups on the surface, as described in Example 3. Figure 8 As shown in C, in the spectrum at 3420 cm⁻¹ -1 and 3450cm -1 It exhibits absorption peaks at 650 cm⁻¹, including the NH stretching vibration of secondary amines and the OH stretching vibration of nitrogen. -1 and 620cm -1 There are CS absorption peaks and sulfite absorption peaks, in the range of 3300-3500 cm⁻¹. -1 There was a distinct broad peak, indicating that the magnetic beads were modified with restriction endonucleases BamHI and HindIII and hydroxyl groups.

[0135] The obtained magnetic beads were used for enzymatic digestion. The substrate and conditions for the enzymatic digestion reaction were the same as in Example 1, and the results are as follows. Figure 7 As shown in well 2, a dragging phenomenon occurs, and star-shaped activity is generated.

[0136] Example 4: An integrated plasmid digestion-recovery kit

[0137] 1. Preparation of plasmid enzyme digestion and recovery reagent

[0138] ① Premixed solution of magnetic bead digestion system (BamHI-HindIII magnetic beads):

[0139] 70 wt% sorbitol solution, 10 mM magnesium acetate (1.42 g / L), 50 mM potassium acetate (4.9 g / L), 20 mM pH 7.9 Tris-acetate (3.27 g / L), 10 μg / ml BSA, 1 wt% magnetic beads prepared in Example 1 (containing 20 U U AmHI and 20 U HindIII);

[0140] ②Enzyme digestion product conjugate: 80% (v / v) isopropanol, 1.5M ammonium acetate, pH 5.5;

[0141] ③ Rinse solution: 80% (v / v) ethanol;

[0142] ④ Eluent: TE solution.

[0143] 2. Plasmid enzymatic digestion and extraction

[0144] 1) Add 100 μL of magnetic bead enzyme digestion system premix, 100 μL of enzyme digestion product binding solution, 500 μL of rinsing solution, and 40 μL of elution solution to wells 1, 2, 3, and 4 of the deep well plate, respectively.

[0145] 2) Take 1 μg of plasmid PET28a and add it to well 1 of a deep-well plate. Digest the plasmid at 37℃ for 15 min, then magnetically aspirate for 1 min. The plasmid PET28a pattern is shown below. Figure 9 As shown;

[0146] 3) Add the reaction product from step 2) into well 2 of the deep well plate, mix well for 2 minutes, and then magnetically aspirate for 1 minute;

[0147] 4) Add the reaction product from step 3) into well 3 of the deep well plate, mix for 1 min, and then magnetically aspirate for 1 min;

[0148] 5) Add the reaction product from step 4) to well 4 of the deep well plate, mix for 2 min, and then magnetically aspirate for 1 min to obtain the plasmid digestion and recovery product.

[0149] The results of agarose gel electrophoresis verification are as follows: Figure 10As shown, lane 1 contains the unpurified and unrecovered enzyme digestion product; lane 2 contains the product obtained in Example 4; lane 3 contains the enzyme digestion product after normal gel extraction and recovery (control); and lane 4 contains the undigested PET28a vector fragment. The initial size was 5369 bp, and the two bands after digestion were 25 bp and 5344 bp, respectively. Since the agarose gel concentration was 1%, only bands larger than 1500 bp could be distinguished; therefore, the gel image shows single bands in wells 1, 2, and 3 after digestion.

[0150] The enzyme digestion products were detected using an ultra-micro spectrophotometer, and the results are shown in Table 1. Sample 1 is the product obtained in Example 4; Sample 2 is the enzyme digestion product after normal gel extraction and recovery (control); A260 represents the absorbance of nucleic acids (DNA and RNA) at a wavelength of 260 nm; A280 represents the absorbance of proteins at a wavelength of 280 nm; 260 / 280 and 260 / 230 represent DNA purity.

[0151] Table 1. Recovery efficiency of enzyme digestion products

[0152] ng / ul A260 A280 260 / 280 260 / 230 Sample 1 12.15 0.243 0.109 2.23 0.18 Sample 2 5.25 0.105 0.046 2.29 0.06

[0153] from Figure 10 As shown in Table 1, the linearized plasmid concentration is basically the same as that obtained using the kit provided by this invention. However, this method shortens the experimental time and reduces the number of steps required. The extraction process of the enzyme digestion product can be completed in 5 minutes using the kit provided by this invention, and the plasmid digestion and recovery process only takes 20 minutes, which is convenient and fast.

[0154] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural changes made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A magnetic bead, characterized in that: The surface of the magnetic beads is modified with restriction endonuclease and hydroxyl groups.

2. The magnetic bead according to claim 1, characterized in that The restriction endonuclease is one or both of BamHI and HindIII. Preferably, the restriction endonucleases are BamHI and HindIII. ， Preferably, the enzyme activity ratio of BamHI modified on the surface of the magnetic beads to HindIII modified on the surface of the magnetic beads is 1:3-3:1, preferably 1:1; Preferably, the particle size of the magnetic beads is 500 nm-1 μm.

3. The method for preparing magnetic beads according to claim 1 or 2, characterized in that: The preparation method of the magnetic beads comprises the following steps: using an activator to activate and modify a restriction endonuclease, and then reacting the activated and modified restriction endonuclease with nano-ferroferric oxide in an alkaline environment to obtain magnetic beads with the restriction endonuclease and hydroxyl groups modified on the surface.

4. The preparation method according to claim 3, characterized in that: The activator is one or a combination of two or more of chloropropyltrimethoxysilane and chloropropyltriethoxysilane. Preferably, the activator is chloropropyltriethoxysilane. and / or, the ratio of the activator to the restriction endonuclease is 1:2-10 in μL / U; And / or, the reaction temperature of the activation modification is 20-30°C; And / or, the reaction time of activation modification is 0.5-2h.

5. The preparation method according to claim 3 or 4, characterized in that: The activated modified restriction endonuclease reacts with nano-ferroferric oxide in a reaction solution with a pH value of 8-9; Preferably, the reaction solution is a Tris reaction solution; Preferably, the reaction temperature is 0-8°C; Preferably, the reaction time is 20-30 hours.

6. The preparation method according to any one of claims 3 to 5, characterized in that: The restriction endonuclease is one or both of BamHI and HindIII. Preferably, the restriction endonuclease is BamHI and HindIII. and / or, the ratio of the activated modified restriction endonuclease to the magnetic beads is 1-4:1 in U / mg; Preferably, the ratio of activated modified BamHI to magnetic beads is 1-3:1 in terms of U / mg; Preferably, the ratio of activated modified HindIII to magnetic beads is 1-3:1 in U / mg.

7. The magnetic beads obtained by the preparation method according to any one of claims 3 to 6.

8. A kit, characterized in that: The kit contains the magnetic beads according to claim 1 or 2 or the magnetic beads according to claim 7; Preferably, the kit contains a magnetic bead enzyme digestion system premix, an enzyme digestion product binding solution, a rinse solution and an eluent; Wherein, the magnetic beads are contained in the premix of the magnetic beads enzyme cleavage system.

9. The kit according to claim 8, characterized in that The magnetic bead enzyme digestion system premix contains Tris-acetate, sorbitol and the magnetic beads; Preferably, the concentration of Tris-acetate in the magnetic bead enzymatic digestion system premix is ​​15-30 mM, preferably 20 mM, preferably, the pH value of Tris-acetate is 7.5-8.5, preferably, the pH value of Tris-acetate is 7.9; Preferably, the concentration of sorbitol in the premix of the magnetic bead enzymatic cleavage system is 60-80wt%, preferably 70wt%. Preferably, the concentration of magnetic beads in the magnetic bead enzymatic cleavage system premix is ​​0.5-2wt%, preferably 1wt%; Preferably, the magnetic bead enzymatic digestion system premix further contains magnesium acetate, potassium acetate and BSA; More preferably, the concentration of magnesium acetate in the magnetic bead enzymatic cleavage system premix is ​​5-15 mM, preferably 10 mM; More preferably, the concentration of potassium acetate in the magnetic bead enzymatic digestion system premix is ​​40-60 mM, preferably 50 mM; More preferably, the concentration of BSA in the magnetic bead enzymatic cleavage system premix is ​​5-15 μg / ml, preferably 10 μg / ml; and / or, the enzyme cleavage product binding solution contains alcohol and salt; Preferably, the volume percentage of alcohol in the enzyme cleavage product binding solution is 75-85%, preferably 80%; Preferably, the alcohol is isopropanol and the salt is ammonium acetate; More preferably, the enzymatic product binding solution contains 1-2M ammonium acetate, preferably 1.5M ammonium acetate; Preferably, the pH value of the enzymatic cleavage product binding solution is 5-6, preferably 5.

5.

10. Use of the magnetic beads according to claim 1 or 2, or the magnetic beads according to claim 7, or the kit according to claim 8 or 9 in plasmid enzyme digestion recovery.

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