Automatic extraction method of endotoxin-free large-volume plasmid DNA and kit

Abstract

The invention relates to the technical field of plasmid extraction, in particular to an automatic extraction method of endotoxin-free large-volume plasmid DNA and a kit. According to the kit provided by the invention, a magnetic bead adsorption technology and a buffer system with a unique separation effect are adopted, and specially modified protein is combined with magnetic beads, so that endotoxin and protein impurities can be adsorbed; an endotoxin removing solution is adopted, so that endotoxin can be efficiently removed; the method comprises the following steps: firstly, preparing plasmid DNA, then, uniquely embedded nucleic acid adsorption magnetic beads have very strong affinity to the plasmid DNA under a certain condition and are not easy to adsorb endotoxin, and when the condition is changed, the nucleic acid adsorption magnetic beads release the adsorbed plasmid DNA, so that the purpose of rapidly separating and purifying the plasmid DNA is achieved. The plasmid DNA extracted by utilizing the kit and the extraction method disclosed by the invention is high in yield, good in purity and extremely low in endotoxin content, and can be suitable for transfecting various cells and various conventional operations including enzyme digestion, PCR (Polymerase Chain Reaction), sequencing, connection and other various downstream experiment requirements.

Description

Technical Field

[0001] The present application relates to the technical field of plasmid extraction, and in particular to an automated extraction method and kit for endotoxin-free large-volume plasmid DNA. Background Art

[0002] The separation and extraction of plasmids is one of the most commonly used and basic experimental techniques in molecular biology. Plasmid DNA with high yield and good purity is an important prerequisite for studying genetic engineering and its upstream and downstream applications. Endotoxin is the main component of the outer membrane of the cell wall of Gram-negative bacteria, also known as lipopolysaccharide. It is released when the bacteria are lysed, which will seriously affect downstream experiments such as the DNA transfection efficiency of primary cells and sensitive cultured cells, and further affect research fields such as nucleic acid vaccines and gene therapy. Therefore, in high-tech research fields such as DNA vaccines and gene therapy, it is particularly critical to extract and purify high-quality plasmid DNA without endotoxin residues.

[0003] At present, the most common plasmid extraction method is the alkaline lysis method based on the traditional principle, which uses the first solution to suspend the bacteria, uses the second solution to perform alkaline lysis, and uses the third solution to separate the plasmid from the bacterial genomic DNA after renaturation, removes proteins and other impurities, and obtains a relatively pure plasmid. The above method is relatively cumbersome and does not specifically remove endotoxins effectively.

[0004] Therefore, it is an urgent need to develop an automated extraction method and kit for large-volume plasmid DNA that is simple to operate, high-quality, and free of endotoxin residues. Summary of the invention

[0005] The present application provides an automated extraction method and kit for endotoxin-free large-volume plasmid DNA.

[0006] The present application aims to address the problems of complex operation and poor removal effect of current endotoxin removal methods in large-volume samples, and to provide an automated extraction method and kit for endotoxin-free large-volume plasmid DNA. The extracted plasmid DNA has a high yield, good purity, and low endotoxin content. At the same time, the operation is simple, and time-saving and labor-saving.

[0007] The kit provided by the present application adopts a unique separation magnetic bead adsorption technology and buffer system, which can separate and purify high-quality plasmid DNA from 50-200ml bacterial solution. The specially modified protein-bound magnetic beads can adsorb endotoxins and protein impurities; the endotoxin removal solution can efficiently remove endotoxins; and then the uniquely embedded nucleic acid adsorption magnetic beads have a strong affinity for plasmid DNA under certain conditions, and are not easy to adsorb endotoxins. When the conditions change, the nucleic acid adsorption magnetic beads release the adsorbed plasmid DNA, thereby achieving the purpose of rapid separation and purification of plasmid DNA.

[0008] In addition, the kit provided by this application is combined with the TGuide LP12 automated biomolecule purification system, which can extract 1-12 sample plasmid DNA at a time. With a single-channel pipette and a 12-channel pipette, the entire process of plasmid extraction from bacterial precipitation to the machine is fully automatically completed. No manual intervention is required during the operation of the all-in-one machine, and the extracted product is automatically stored in the collection tube, which is simple to operate and saves time and effort. At the same time, the plasmid DNA extracted using the kit and extraction method of this application has a high yield, good purity, and extremely low endotoxin content, which can be applied to transfect a variety of cells and various routine operations, including enzyme cutting, PCR, sequencing, ligation and other downstream experimental needs.

[0009] In the first aspect, the present application provides an automated extraction kit for large-volume plasmid DNA without endotoxin, which adopts the following technical scheme:

[0010] An automated extraction kit for endotoxin-free large-volume plasmid DNA, the kit comprising the following components: a lysis solution, an endotoxin removal solution, an isopropanol solution, an impurity removal solution, an elution solution, and magnetic beads;

[0011] The endotoxin removal solution includes 10-30% by volume of Triton X-114; 0.001-0.005% by mass volume of phenol red; 0.2-1% by mass volume of sodium deoxycholate; and sodium acetate with a concentration of 0.2-0.8M and a pH of 5.5.

[0012] The magnetic bead particles are any one or more of silicon hydroxy magnetic beads and silicon carboxyl magnetic beads; including protein binding magnetic beads for binding endotoxins and protein impurities and nucleic acid adsorption magnetic beads for binding plasmid DNA obtained by separation and purification, and the particle size range is 200nm-3μm.

[0013] Optionally, the protein-binding magnetic beads are a mixture of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads.

[0014] Optionally, the mixing ratio of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads in the protein-binding magnetic beads is (3-1):1.

[0015] Optionally, the mixing ratio of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads in the protein-binding magnetic beads is 2:1.

[0016] Optionally, the particle size of the protein-bound magnetic beads ranges from 400 nm to 3 μm.

[0017] Optionally, the nucleic acid adsorbing magnetic beads are silica-carboxyl magnetic beads.

[0018] Optionally, the particle size range of the nucleic acid adsorption magnetic beads is 400 nm.

[0019] In the present application, the endotoxin removal solution can specifically bind to the endotoxin released after the bacterial cells are broken. The magnetic bead particles are any one or more of silicon hydroxy magnetic beads and silicon carboxyl magnetic beads, including protein-binding magnetic beads and nucleic acid-absorbed magnetic beads, with a particle size range of 200nm-3μm. Protein-binding magnetic beads are used to bind endotoxins and protein impurities, while nucleic acid-absorbed magnetic beads are used to bind plasmid DNA obtained by separation and purification.

[0020] Optionally, the volume percentage of the isopropanol solution is 20-40%.

[0021] In the present application, adding a certain proportion of isopropanol solution to the environment of the endotoxin removal solution can further effectively reduce the endotoxin residue in the plasmid DNA.

[0022] Optionally, the lysis solution includes a bacterial resuspension solution, an alkaline lysis solution and a neutralization solution.

[0023] Optionally, the bacterial resuspension solution includes Tris-Cl buffer with a pH of 7.5-8.0 and a concentration of 20-50 mM; EDTA with a pH of 8.0, 25° C. and a concentration of 20-50 mM; RNase A with a concentration of 50-100 mg / ml; and lysozyme with a concentration of 20-80 ng / ml.

[0024] Optionally, the alkaline lysis solution comprises NaOH with a concentration of 100-200 mM, SDS with a mass volume ratio of 1-2%, and isopropanol with a volume percentage of 5-10%.

[0025] Optionally, the neutralization solution comprises potassium acetate with a pH of 3.5-4.0 and a concentration of 2-4M.

[0026] Optionally, the impurity removal solution includes a protein removal solution and a rinsing solution.

[0027] Optionally, the protein removal solution includes: guanidine hydrochloride at a concentration of 1-5M; sodium acetate at a pH of 3.5-4.0 and a concentration of 1-5M; Triton X-114 at a volume percentage of 10-20%; sodium deoxycholate at a volume percentage of 0.5-1%; PEG4000 at a volume percentage of 10-20%; sodium chloride at a concentration of 2-5M; and anhydrous ethanol at a volume percentage of 40-60%.

[0028] Optionally, the rinsing solution includes: Tris with a pH of 5.5-6.0 and a concentration of 1-2 M; and anhydrous ethanol with a volume percentage of 40-60%.

[0029] In the present application, the protein removal solution includes components such as guanidine hydrochloride, Triton X-114, sodium deoxycholate, etc., which can more effectively remove protein impurities, endotoxins and large genomic fragments attached to the magnetic beads. The rinse solution includes Tris and anhydrous ethanol, which can further remove salt ions bound to the magnetic beads.

[0030] Optionally, the elution solution is nuclease-free water.

[0031] In a second aspect, the present application provides an automated extraction method for endotoxin-free large-volume plasmid DNA. The automated extraction method uses the automated extraction kit described above in combination with an automated biomolecule purification system to extract plasmid DNA from a sample.

[0032] Optionally, the automated biomolecule purification system is a TGuide LP12 automated biomolecule purification system;

[0033] Optionally, the automated extraction method can extract plasmid DNA from 1 to 12 samples at one time.

[0034] The kit provided in this application is used to extract plasmid DNA from the sample, and the endotoxin content of the extracted plasmid DNA is less than 20EU / mg. The kit provided in this application is combined with the TGuide LP12 automated biomolecule purification system, which can automatically complete the entire process of plasmid extraction from bacterial precipitation to the machine. No manual intervention is required during the operation of the all-in-one machine, and the extracted product is automatically stored in the collection tube, which is simple to operate and saves time and effort.

[0035] When the above-mentioned automated extraction kit is combined with an automated biomolecule purification system to extract plasmid DNA from a sample, the following steps are included:

[0036] (1) The alkaline lysis method is used, using a bacterial resuspension solution and an alkaline lysis solution. After the bacterial cells are resuspended, the cells are gently lysed, and then the residual alkaline lysis solution is neutralized with a neutralizing solution. The proteins are entangled with each other by rotating the centrifuge tube, thereby tightly adsorbing endotoxins and protein impurities on the protein-bound magnetic beads.

[0037] (2) In the environment of endotoxin removal solution, the nucleic acid adsorption magnetic beads have a strong affinity for plasmid DNA, binding the plasmid DNA to the nucleic acid adsorption magnetic beads, and are not easy to adsorb endotoxin.

[0038] (3) Protein impurities, residual endotoxins, and bound genomic DNA are washed away using a protein removal solution.

[0039] (4) Add rinse solution to remove residual salt ions.

[0040] (5) adding an elution solution and changing the conditions to release the adsorbed plasmid DNA from the nucleic acid adsorption magnetic beads, thereby achieving the purpose of rapid separation and purification of the plasmid DNA.

[0041] After experimental analysis, the plasmid DNA extracted using the kit of the present application has a high yield, good purity, and extremely low endotoxin content, and can be suitable for transfection of a variety of cells and various routine operations, including enzyme digestion, PCR, sequencing, ligation and other downstream experimental needs.

[0042] In a specific embodiment, the above-mentioned automated extraction kit is combined with an automated biomolecule purification system to extract plasmid DNA from a sample, comprising the following steps:

[0043] (1) sequentially adding bacterial resuspension solution, alkaline lysis solution and neutralization solution to the sample to be tested;

[0044] (2) Add protein-binding magnetic beads to tightly adsorb protein impurities;

[0045] (3) Adsorbed by magnetic rack, and the supernatant was collected;

[0046] (4) adding endotoxin removal solution, isopropanol solution and nucleic acid adsorption magnetic beads in sequence to tightly bind the plasmid DNA to the nucleic acid adsorption magnetic beads;

[0047] (5) adding protein removal solution for rinsing;

[0048] (6) adding rinsing liquid for rinsing;

[0049] (7) Adding elution solution, the nucleic acid adsorption magnetic beads release the adsorbed plasmid DNA to obtain separated and purified plasmid DNA.

[0050] In summary, the present application includes at least one of the following beneficial technical effects:

[0051] The kit provided in the present application uses specially modified protein-bound magnetic beads to adsorb endotoxins and protein impurities; an endotoxin removal solution is used to efficiently remove endotoxins; then, the uniquely embedded nucleic acid adsorption magnetic beads have a strong affinity for plasmid DNA under certain conditions and are not easy to adsorb endotoxins; the protein removal solution is used to wash away protein impurities, residual endotoxins and bound genomic DNA; the rinse solution washes away residual salt ions; and when the conditions change, the nucleic acid adsorption magnetic beads release the adsorbed plasmid DNA, thereby achieving the purpose of rapid separation and purification of plasmid DNA, thereby ensuring the purity of the extracted plasmid and the residual endotoxins.

[0052] In addition, the kit provided in this application is combined with the TGuide LP12 automated biomolecule purification system to extract plasmid DNA from 1 to 12 samples at a time. The samples do not require any pretreatment, and the entire process of plasmid extraction from bacterial precipitation to the machine is completed automatically. No manual intervention is required during the operation of the all-in-one machine, and the extracted products are automatically stored in the collection tube, which is simple to operate and saves time and effort.

[0053] At the same time, the plasmid DNA extracted using the kit and extraction method of the present application has a high yield, good purity, and extremely low endotoxin content, and can be suitable for transfection of a variety of cells and various routine operations, including enzyme digestion, PCR, sequencing, ligation and other downstream experimental needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the TGuide LP12 operating table

[0055] Figure 2 The results of plasmid DNA extraction using differently modified protein-bound magnetic beads (Preparation Examples 1-9).

[0056] Figure 3 The results of plasmid DNA extraction using protein-bound magnetic beads at different mixing ratios (Preparation Example 1, Preparation Examples 10-11).

[0057] Figure 4 The results of plasmid DNA extraction using differently modified nucleic acid adsorption magnetic beads (Preparation Examples 12-19).

[0058] Figure 5 The results of plasmid DNA extraction using protein removal solutions of different components (Preparation Examples 20-26).

[0059] Figure 6 The figures are the extraction results of plasmid DNA in the embodiments and different comparative examples.

[0060] Figure 7 Extraction results of plasmid DNA extracted for the TGuide LP12 automated biomolecule purification system. DETAILED DESCRIPTION

[0061] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0062] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0063] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0064] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0065] The present application provides an automated extraction kit for large-volume plasmid DNA without endotoxin, which specifically includes the following components: a lysis solution (including a bacterial resuspension solution, an alkaline lysis solution and a neutralization solution), protein-bound magnetic beads, an isopropanol solution, nucleic acid adsorption magnetic beads, an endotoxin removal solution, an impurity removal solution (including a protein removal solution and a rinse solution), and an elution solution.

[0066] The bacterial resuspension solution includes Tris-Cl buffer with a pH of 7.5-8.0 and a concentration of 20-50 mM; EDTA with a pH of 8.0, 25° C. and a concentration of 20-50 mM; RNase A with a concentration of 50-100 mg / ml; and lysozyme with a concentration of 20-80 ng / ml.

[0067] The alkaline lysis solution includes NaOH with a concentration of 100-200 mM, SDS with a mass volume ratio of 1-2%, and isopropanol with a volume percentage of 5-10%.

[0068] The neutralization solution includes potassium acetate with a pH of 3.5-4.0 and a concentration of 2-4M.

[0069] The protein-bound magnetic beads are any one or more of silicon hydroxy magnetic beads and silicon carboxyl magnetic beads, with a particle size range of 200nm-3μm, a concentration of 100mg / ml, and a single use volume of 2.5-4mL.

[0070] Among them, the nucleic acid adsorption magnetic beads are silica carboxyl magnetic beads with a particle size range of 200nm-500nm, a concentration of 50mg / ml, and a single use volume of 300-600μL.

[0071] The endotoxin removal solution includes 10-30% Triton X-114 by volume; 0.001-0.005% phenol red by mass volume; 0.2-1% sodium deoxycholate by mass volume; and sodium acetate with a concentration of 0.2-0.8M and a pH of 5.5.

[0072] The volume percentage of the isopropanol solution is 20-40%.

[0073] The protein removal solution includes: guanidine hydrochloride with a concentration of 1-5M; sodium acetate with a pH of 3.5-4.0 and a concentration of 1-5M; Triton X-114 with a volume percentage of 10-20%; sodium deoxycholate with a volume percentage of 0.5-1%; PEG4000 with a volume percentage of 10-20%; sodium chloride with a concentration of 2-5M; and anhydrous ethanol with a volume percentage of 40-60%.

[0074] The rinse solution includes: Tris with a pH of 5.5-6.0 and a concentration of 1-2M; and anhydrous ethanol with a volume percentage of 40-60%.

[0075] The elution solution is nuclease-free water.

[0076] When the above-mentioned automated extraction kit is combined with an automated biomolecule purification system to extract plasmid DNA from a sample, the following steps are included:

[0077] (1) Pretreatment of bacterial sample

[0078] Take 50-200ml of overnight cultured bacterial solution and add it to 50ml centrifuge tubes in batches. Centrifuge at 8,000rpm (~13,400×g) for 3min and remove the supernatant as much as possible. If more than 50ml of bacterial solution is enriched, the bacterial precipitate can be collected into a centrifuge tube by several centrifugations. The amount of bacterial cells should be sufficient to lyse. Excessive bacterial lysis will reduce the efficiency of plasmid extraction. If it is a bacterial sludge sample, directly weigh about 0.5-1.5g of bacterial sludge and put it into a 50ml centrifuge tube.

[0079] (2) Add 9 ml of bacterial resuspension solution to the centrifuge tube containing the bacterial cell pellet and use a pipette or vortex oscillator to thoroughly resuspend the bacterial cell pellet.

[0080] (3) Add 9 ml of alkaline lysis solution to the centrifuge tube and gently invert it upside down 6-8 times to fully lyse the bacteria.

[0081] (4) Add 9 ml of neutralization solution to the centrifuge tube and immediately and gently invert it upside down 6-8 times to mix thoroughly. A white flocculent precipitate will appear and then leave it at room temperature for about 10 minutes.

[0082] (5) Add 3 ml of protein-bound magnetic beads that have been thoroughly mixed in advance to the centrifuge tube and turn it upside down for 5 minutes. After thorough mixing, place the centrifuge tube on the magnetic rack and leave it for 5 minutes. When the magnetic beads are completely adsorbed, take 21 ml of the supernatant and transfer it to a new centrifuge tube.

[0083] (6) Add 6 ml of endotoxin removal solution to the supernatant and mix by inverting until the solution becomes a uniform, transparent yellow color.

[0084] (7) Add 9 ml of isopropanol to the supernatant and mix thoroughly by inverting.

[0085] (8) Add 300 μl of pre-mixed nucleic acid adsorption magnetic beads to the mixed solution, mix by inversion for 5 minutes, and then place the centrifuge tube on the magnetic rack for 2 minutes. When the magnetic beads are completely adsorbed, discard the supernatant.

[0086] (9) Remove the centrifuge tube from the magnetic rack, add 30 ml of protein removal solution, mix by inversion for 5 minutes, and then place the centrifuge tube on the magnetic rack for 2 minutes. When the magnetic beads are completely adsorbed, discard the supernatant.

[0087] (10) Remove the centrifuge tube from the magnetic rack, add 6 ml of protein removal solution, mix by inversion for 5 minutes, and then place the centrifuge tube on the magnetic rack for 2 minutes. When the magnetic beads are completely adsorbed, discard the supernatant.

[0088] (11) Remove the centrifuge tube from the magnetic rack, add 30 ml of rinse solution, mix by inversion for 5 minutes, and then place the centrifuge tube on the magnetic rack for 2 minutes. When the magnetic beads are completely adsorbed, discard the supernatant.

[0089] (12) Remove the centrifuge tube from the magnetic rack, add 6 ml of rinse solution, mix by inverting for 5 minutes, and then place the centrifuge tube on the magnetic rack for 2 minutes. When the magnetic beads are completely adsorbed, discard the supernatant. Remove as much liquid as possible.

[0090] (13) Place the centrifuge tube on a magnetic rack and let it dry at room temperature for 5-10 min.

[0091] (14) Remove the centrifuge tube from the magnetic rack, add 1 ml of elution buffer, and leave at room temperature for 5 min, shaking gently several times.

[0092] (15) Place the centrifuge tube on a magnetic rack and let it stand for 2 min. When the magnetic beads are completely adsorbed, carefully transfer the plasmid DNA to a 1.5 ml centrifuge tube and store it at -30 to -15°C.

[0093] The solution in the kit of this application can be used with the TGuide LP12 automated biomolecule purification system to extract 1-12 samples at a time. The samples do not require any pre-treatment. The centrifuged bacteria are directly placed in the "sample area" of the TGuide LP12 instrument after removing the tube cap. The remaining reagents and consumables used, such as 10ml pipette tips, 50ml centrifuge tubes, 1.5ml collection tubes, etc., are placed in the required quantities in the various wells of the reagent area on the left side of the TGuide LP12 instrument operating table (refer to Table 1 and Table 2 for the corresponding positions and reagents). Figure 1 ). Follow the machine operating instructions and select the plasmid extraction program corresponding to the sample quantity (the program is edited according to the above steps). The final extracted plasmid DNA will be automatically stored in a 1.5ml collection tube and stored under appropriate conditions.

[0094] Table 1 TGuide LP12 reagent area placement guide

[0095] Reagent placement Reagent name Reagent placement Reagent name Reagent well 1 Protein Removal Solution Reagent well 6 Protein-binding magnetic beads Reagent well 2 Isopropyl alcohol solution Reagent well 7 Bacteria resuspension solution Reagent well 3 Endotoxin Removal Solution Reagent well 8 Alkaline lysis solution Reagent well 4 Eluent Reagent well 9 neutralizing solution Reagent well 5 Rinse liquid Reagent well 10 Nucleic acid adsorption magnetic beads

[0096] In this application, the protein-binding magnetic beads are a mixture of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads in a mass ratio of 2:1. The particle size of silicon carboxyl magnetic beads is 400nm, purchased from Suzhou Weidu, with the item number MA0309C; the particle size of silicon hydroxyl magnetic beads is 3μm, purchased from TIANGEN, with the item number MG101. The nucleic acid adsorption magnetic beads are silicon carboxyl magnetic beads with a particle size of 400nm, purchased from Jinzhun Zhizao, with the item number 50102.

[0097] The remaining raw materials, reagents, solvents, etc. of this application can also be obtained through commercial purchase.

[0098] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0099] If no specific techniques or conditions are specified in the preparation examples and examples, the preparations were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0100] The present application is further described in detail below in combination with preparation examples, embodiments, drawings and test results.

[0101] Preparation Example

[0102] Preparation Example 1

[0103] This preparation example provides an automated extraction kit for large-volume plasmid DNA without endotoxin.

[0104] The above-mentioned kit includes lysis solution (including bacterial resuspension solution, alkaline lysis solution and neutralization solution), protein-bound magnetic beads, isopropanol solution, nucleic acid adsorption magnetic beads, endotoxin removal solution, impurity removal solution (including protein removal solution and rinsing solution), and elution solution.

[0105] Among them, the bacterial resuspension solution, alkaline lysis solution, neutralization solution, protein-binding magnetic beads, isopropanol solution, nucleic acid adsorption magnetic beads, endotoxin removal solution, protein removal solution and rinsing solution are all aqueous solutions prepared with water as solvent.

[0106] The components and contents of the above solutions are shown in Table 2.

[0107] Table 2 Components and contents of each solution in the large volume endotoxin-free plasmid extraction kit

[0108]

[0109] Preparation Example 2-11

[0110] Preparation Examples 2-11 respectively provide an automated extraction kit for large-volume plasmid DNA without endotoxin.

[0111] The difference between the above preparation example and preparation example 1 is that the protein-bound magnetic beads are different, as shown in Table 3. The remaining components are the same as those in preparation example 1.

[0112] The details are as follows:

[0113] The difference between Preparation Example 1-9 is that the protein-bound magnetic beads are replaced with magnetic beads of other brands or different modified types.

[0114] The difference between Preparation Examples 1 and 10-11 lies in the mixing ratio of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads in the protein-binding magnetic beads.

[0115] Table 3 Types of protein-bound magnetic beads in Preparation Example 2-11

[0116] Preparation Example Protein-binding magnetic beads Particle size 2 Silicon Hydroxyl Magnetic Beads (TIANGEN) 3μm 3 Silica carboxyl magnetic beads (Aisen Biotechnology) 500nm 4 Silica carboxyl magnetic beads (Aisen Biotechnology) 300nm 5 Amino magnetic beads (Jike Biotechnology) 400nm 6 PS magnetic beads (Qiyue Biotechnology) 1μm 7 Silicon carboxyl magnetic beads (Jinzhun Intelligent Manufacturing) 400nm 8 Silicon carboxyl magnetic beads (Jinzhun Intelligent Manufacturing) 50nm 9 Silicon carboxyl magnetic beads (Jinzhun Intelligent Manufacturing) 200nm 10 Mixture of Silane Carboxyl Magnetic Beads and Silane Hydroxyl Magnetic Beads (mixing ratio 3:1) 400nm-3μm 11 Mixture of Silane Carboxyl Magnetic Beads and Silane Hydroxyl Magnetic Beads (mixing ratio 1:1) 400nm-3μm

[0117] Preparation Examples 12-19

[0118] Preparation Examples 12-19 respectively provide an automated extraction kit for large-volume plasmid DNA without endotoxin.

[0119] The difference between the above preparation example and preparation example 1 is that the nucleic acid adsorption magnetic beads are replaced with magnetic beads of other brands or different modification types, as shown in the following Table 4. The remaining components are the same as those in preparation example 1.

[0120] Table 4 Types of nucleic acid adsorption magnetic beads in Preparation Examples 12-19

[0121]

[0122]

[0123] Preparation Examples 20-26

[0124] Preparation Examples 20-26 respectively provide an automated extraction kit for large-volume plasmid DNA without endotoxin.

[0125] The difference between the above preparation example and preparation example 1 is that the components of the protein removal solution are adjusted, as shown in Table 5. The remaining components are the same as those in preparation example 1.

[0126] Table 5 Components of the protein removal solution in Preparation Example 1 and Preparation Examples 20-26

[0127]

[0128] Preparation Examples 27-28

[0129] Preparation Examples 27-28 respectively provide an automated extraction kit for large-volume plasmid DNA without endotoxin.

[0130] The difference between the above preparation example and preparation example 1 is that the proportion of anhydrous ethanol in the rinsing liquid is adjusted, as shown in Table 6. The remaining components are the same as those in preparation example 1.

[0131] Table 6 Proportion of anhydrous ethanol in the rinse solution in Preparation Example 1 and Preparation Examples 27-28

[0132]

[0133] Examples 1-28

[0134] Examples 1-28 each provide an automated method for extracting large-volume plasmid DNA without endotoxin.

[0135] The above examples respectively used the kits provided in the above preparation examples 1-28 to perform automated extraction of plasmids.

[0136] The above-mentioned automated extraction method specifically comprises the following steps:

[0137] (1) Pretreatment of bacterial liquid samples: Take 150 ml of overnight cultured AAV E. coli liquid and add it to a 50 ml centrifuge tube in batches. Centrifuge at 8,000 rpm (~13,400 × g) for 3 min and remove the supernatant as much as possible. If it is a bacterial sludge sample, directly weigh about 1.5 g of bacterial sludge and put it into a 50 ml centrifuge tube.

[0138] (2) Reagent preparation and packaging: Prepare each reagent according to Preparation Examples 1-26 and package them into reagent bottles of corresponding volumes.

[0139] (3) TGuide LP12 automated biomolecule purification system operation steps:

[0140] Remove the tube cap after centrifugation and place it in the "sample area" of the TGuide LP12 instrument. Place the remaining reagents and consumables such as 10ml pipette tips, 50ml centrifuge tubes, 1.5ml collection tubes, etc. into the wells of the reagent area on the left side of the TGuide LP12 instrument operating table according to the required quantity (refer to Table 1 and Table 2 for the corresponding positions and reagents). Figure 1 ).

[0141] Follow the machine's operating instructions and select the plasmid extraction program corresponding to the sample quantity. The final extracted plasmid DNA will be automatically stored in a 1.5ml collection tube and stored under appropriate conditions.

[0142] Examples 29-32

[0143] Examples 29-32 each provide an automated method for extracting large-volume plasmid DNA without endotoxin.

[0144] The difference between the above embodiment and embodiment 1 is that in the automated extraction method, the content of the isopropanol solution is adjusted, as shown in Table 7. The remaining steps are consistent with those in embodiment 1.

[0145] Table 7 Content of isopropanol solution of Example 1 and Examples 29-32

[0146] Example Isopropanol solution (ml) Corresponding isopropanol concentration in the system 1 9 30％ 29 11 37％ 30 10 33％ 31 8 27％ 32 7 23％

[0147] Comparative Example

[0148] Comparative Example 1

[0149] This comparative example provides a method for extracting plasmids using a commercially available endotoxin-free plasmid extraction kit. The endotoxin-free plasmid extraction kit is a centrifugal column type, purchased from TIANGEN, item number: DP117.

[0150] Comparative Example 2

[0151] This comparative example provides a method for extracting plasmids using a commercially available endotoxin-free plasmid extraction kit. The endotoxin-free plasmid extraction kit is an enhanced type, purchased from TIANGEN, item number: DP120.

[0152] Comparative Example 3

[0153] This comparative example provides a method for extracting plasmids using a commercially available endotoxin-free plasmid extraction kit. The endotoxin-free plasmid extraction kit is Easy Endo Free Pro Plasmid MaxiPrep Kit, purchased from TransGen, catalog number: EM123.

[0154] Test result 1

[0155] The plasmid DNA extracted from Examples 1-32 and Comparative Examples 1-3 were tested respectively.

[0156] The concentration of plasmid DNA was determined by NanoDrop 2000 and detected by agarose electrophoresis.

[0157] The endotoxin content of the extracted plasmid DNA was determined using an endotoxin detection horseshoe crab kit (test tube quantitative chromogenic matrix method). The specific operation method is shown in the kit manual. The kit was purchased from Xiamen Horseshoe Crab Reagent Co., Ltd., item number EC80545S.

[0158] The test results are shown in Table 8, and the agarose electrophoresis results are shown in Table 8. Figure 2-6 shown.

[0159] Table 8 Detection results of plasmid DNA extracted from Examples 1-32 and Comparative Examples 1-3

[0160]

[0161]

[0162] According to Table 8 and Figure 2-6 It can be seen from the test results of Examples 1-11 that the protein-bound magnetic beads of Example 6 have a higher yield and a stronger adsorption effect on endotoxins, but in the actual operation process, the protein adsorption is relatively loose, and the protein is not easy to adsorb on the magnetic frame, and the experiment is very accidental. The protein-bound magnetic beads of Example 5 have a lower yield, and the protein adsorption effect is not good, and the endotoxin residue is also more serious. The magnetic beads of Examples 7, 8 and 9 are conducive to the extraction of plasmids in large volumes, but white precipitates are easily produced in the eluent. The remaining magnetic beads are conducive to plasmid extraction, but the adsorption effect on endotoxins is not strong, and there are different degrees of protein residues. At the same time, the ratio of the mixed solution of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads is adjusted, which is conducive to the extraction of plasmids and the adsorption of proteins and endotoxins. Based on the overall consideration of comprehensive factors such as the magnetic absorption speed and production cost of the protein-bound magnetic beads, the protein-bound magnetic bead mixture used in this kit is preferred.

[0163] It can be seen from the test results of Example 1 and Examples 12-19 that the nucleic acid adsorption magnetic beads of Example 18 have the highest yield, but the residual endotoxin content is also the most serious. In addition, the residual endotoxin content of Examples 17 and 19 is also relatively serious. The extraction efficiency of the plasmid in Example 12 is relatively low. Although the yields of Examples 14 and 16 are relatively high, the residual endotoxin is also relatively high, and there is also color residue in the eluent. The remaining magnetic beads have different degrees of adsorption effect on endotoxin. Therefore, the comprehensive evaluation gives priority to the nucleic acid adsorption magnetic beads used in this kit.

[0164] It can be seen from the test results of Example 1 and Examples 20-26 that different protein removal solutions have a great influence on the yield, ratio and endotoxin residue of the plasmid. After rinsing with the protein removal solution of Examples 24 and 26, a large amount of plasmid loss was caused. The other embodiments also had different degrees of loss in the yield of the plasmid. After rinsing in Examples 20, 21 and 23, there was a certain loss in the yield of the plasmid, but there was not a very good effect on the removal of endotoxin. The remaining protein removal solutions had a comparable yield of the plasmid, but there was still a certain residue in the removal of endotoxin. Considering all factors, the reagent components of this kit were preferentially selected as the protein removal solution.

[0165] It can be seen from the test results of Example 1 and Examples 27-28 that the proportion of anhydrous ethanol in the rinse solution has a great influence on the yield, ratio and endotoxin residue of the plasmid. Too low an alcohol ratio greatly reduces the binding of the nucleic acid adsorption magnetic beads to the plasmid DNA, thereby causing the loss of plasmid DNA during rinsing and a poor ratio; and when the alcohol ratio is too high, although a high plasmid DNA can be obtained, the adsorption of endotoxin is also greatly enhanced. Therefore, selecting an appropriate alcohol ratio can more effectively ensure the plasmid yield while reducing the endotoxin content.

[0166] It can be seen from the test results of Example 1 and Examples 29-32 that the effects of different ratios of isopropanol and endotoxin removal solution are different. Too low an alcohol ratio causes a large loss in the yield of the plasmid and also affects the adsorption effect of the endotoxin removal solution, while a high alcohol ratio also has a certain effect on the adsorption of endotoxin. Therefore, after comprehensive consideration, it is preferred to add 9 ml of isopropanol solution as the required ratio of this kit.

[0167] It can be seen from the test results of Example 1 and Comparative Examples 1-3 that, although the overall plasmid DNA yields are comparable, the endotoxin residues are quite low after the extraction scheme of the kit, which is a significant advantage. In addition, the scheme is combined with an automated biomolecule purification system, and no manual intervention is required during operation, saving time and effort.

[0168] Test result 2

[0169] The kit provided in Example 1 was used to extract plasmid DNA using the TGuide LP12 automated biomolecule purification system. The extraction object was 100 ml of AAV Escherichia coli culture that had been shaken overnight, and 12 parallel samples were set up.

[0170] NanoDrop 2000 was used to measure the concentration of plasmid DNA and agarose electrophoresis, and an endotoxin detection horseshoe crab kit (test tube quantitative chromogenic matrix method) was used to determine the endotoxin content of the extracted plasmid DNA.

[0171] The test results are shown in Table 9, and the agarose electrophoresis results are shown in Table 9. Figure 7 shown.

[0172] Table 9 Extraction results of TGuide LP12 automated biomolecule purification system

[0173] parallel Concentration (ng / μL) A260 / A280 A260 / A230 Endotoxin content (EU / mg) 1 1502.6 1.91 2.17 18.43 2 1688.1 1.91 2.15 21.13 3 1593.5 1.91 2.19 10.05 4 1349.2 1.91 2.23 21.72 5 1632.4 1.9 2.23 15.00 6 1444.6 1.91 2.19 19.79 7 1724 1.91 2.29 15.42 8 1763.9 1.91 2.29 15.76 9 1507.2 1.92 2.14 16.75 10 1543.7 1.92 2.13 16.11 11 1449.5 1.92 2.19 15.98 12 1635.6 1.93 2.16 12.94 average value 1569.53 - - 16.59 STD 119.79 - - 3.19 CV value (%) 7.63 - - 19.21 Average total yield (mg) 1.57 - - -

[0174] Depend on Figure 7 As can be seen from the test results in Table 9, the use of the TGuide LP12 automated biomolecule purification system in conjunction with the large-volume endotoxin-free plasmid extraction kit of the present application only requires simple pre-treatment of the bacterial solution sample, and the rest can be achieved on an automated instrument. 100ml AAV bacterial solution was used to extract plasmid DNA, and the CV value of the yield of 12 wells was 7.63%, with good uniformity. The average plasmid yield per 100ml of bacterial solution can reach 1.57mg. The overall endotoxin residue is low, with a CV value of 19.21%, which is comparable to the results of ordinary large-volume plasmid extractions and can meet downstream experimental needs. It can also save labor, is easy to operate, and achieve high-throughput automated extraction.

[0175] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An automated extraction kit for endotoxin-free large-volume plasmid DNA, characterized in that: The kit comprises the following components: a lysis solution, an endotoxin removal solution, an isopropanol solution, an impurity removal solution, an elution solution, and magnetic beads; The endotoxin removal solution includes 10-30% by volume of Triton X-114; Phenol red at a mass volume ratio of 0.001-0.005%; sodium deoxycholate at a mass volume ratio of 0.2-1%; sodium acetate at a concentration of 0.2-0.8M and a pH of 5.5; The magnetic bead particles are any one or more of silicon hydroxy magnetic beads and silicon carboxyl magnetic beads; including protein binding magnetic beads for binding endotoxins and protein impurities and nucleic acid adsorption magnetic beads for binding plasmid DNA obtained by separation and purification, and the particle size range is 200nm-3µm.

2. The automated extraction kit according to claim 1, characterized in that The volume percentage of the isopropanol solution is 20-40%.

3. The automated extraction kit according to claim 1, characterized in that The protein-binding magnetic beads are a mixture of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads; Optionally, the nucleic acid adsorption magnetic beads are silica carboxyl magnetic beads; Optionally, the mixing ratio of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads in the protein-binding magnetic beads is (3-1):1; Optionally, the mixing ratio of silicon carboxyl magnetic beads and silicon hydroxyl magnetic beads in the protein-binding magnetic beads is 2:

1.

4. The automated extraction kit according to claim 1, characterized in that The lysis solution comprises a bacterial resuspension solution, an alkaline lysis solution and a neutralization solution.

5. The automated extraction kit according to claim 4, characterized in that The bacterial resuspension solution includes a Tris-Cl buffer solution with a pH of 7.5-8.0 and a concentration of 20-50 mM; EDTA with a pH of 8.0, 25° C. and a concentration of 20-50 mM; RNase A with a concentration of 50-100 mg / ml; and lysozyme with a concentration of 20-80 ng / ml. Optionally, the alkaline lysis solution comprises NaOH with a concentration of 100-200 mM, SDS with a mass volume ratio of 1-2%, and isopropanol with a volume percentage of 5-10%; Optionally, the neutralization solution comprises potassium acetate with a pH of 3.5-4.0 and a concentration of 2-4M.

6. The automated extraction kit according to claim 1, characterized in that The impurity removal solution includes a protein removal solution and a rinsing solution.

7. The automated extraction kit according to claim 6, characterized in that The protein removal solution comprises: guanidine hydrochloride with a concentration of 1-5M; sodium acetate with a pH of 3.5-4.0 and a concentration of 1-5M; Triton X-114 with a volume percentage of 10-20%; sodium deoxycholate with a volume percentage of 0.5-1%; PEG4000 with a volume percentage of 10-20%; sodium chloride with a concentration of 2-5M; and anhydrous ethanol with a volume percentage of 40-60%. Optionally, the rinsing solution includes: Tris with a pH of 5.5-6.0 and a concentration of 1-2 M; and anhydrous ethanol with a volume percentage of 40-60%.

8. The automated extraction kit according to claim 1, characterized in that The elution solution is nuclease-free water.

9. An automated method for extracting endotoxin-free large-volume plasmid DNA, characterized in that: The plasmid DNA in the sample is extracted using the automated extraction kit according to any one of claims 1 to 8 in combination with an automated biomolecule purification system.

10. The automated extraction method according to claim 9, characterized in that The automated biomolecule purification system is a TGuide LP12 automated biomolecule purification system; Optionally, the automated extraction method can extract plasmid DNA from 1 to 12 samples at one time.

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