A method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights and its application

Abstract

The application provides a method for detecting the purity of supercoiled DNA in plasmid samples with different molecular weights and application thereof, and comprises the following steps: preparing linearized plasmid samples and untreated plasmid samples for HPLC detection; performing HPLC detection by using an anion exchange column; collecting chromatograms of supercoiled plasmid DNA, open circle plasmid DNA, linear plasmid DNA and concatemer plasmid DNA in linearized and untreated plasmid samples with different molecular weights; and the mobile phase of the HPLC detection comprises: mobile phase A is a water solution containing tris-hydroxymethyl aminomethane, and mobile phase B is a water solution containing tris-hydroxymethyl aminomethane and sodium chloride. The method can ensure complete separation of supercoiled plasmid DNA, open circle plasmid DNA, linear plasmid DNA and concatemer plasmid DNA in plasmid samples with different molecular weights, has high resolution and high sensitivity, and has important application value in the field of nucleic acid drug analysis technology.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid analysis and detection, specifically relating to a method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights and its application, and particularly to a high-resolution, high-sensitivity ion exchange chromatography method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights and its application. Background Technology

[0002] Plasmids are small, self-replicating DNA molecules. Artificially modified plasmids can be used as drugs to express inserted target genes in patients, ultimately aiming to prevent or treat related diseases. Currently, plasmid DNA has become a representative of next-generation biopharmaceuticals, with potential applications in gene vaccines and gene therapy.

[0003] As a key raw material for gene vaccines and gene therapy, the quality of plasmid products has received widespread attention. In plasmid drug analysis, plasmids with different topological isomorphic forms mainly include supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA, and multiplicative plasmid DNA. Some researchers have reported that supercoiled plasmid DNA has higher in vivo transfection efficiency. Therefore, technical guidelines or product-related standards (such as EP, the Pharmacopoeia of the People's Republic of China, and USP) issued by domestic and international drug regulatory agencies (such as the FDA and EMA) related to gene therapy products all include the content of supercoiled plasmid DNA as one of the quality control requirements for plasmid DNA purity.

[0004] However, existing methods for detecting supercoiled plasmid DNA all have certain limitations. Agarose gel electrophoresis has poor reproducibility and accuracy; capillary electrophoresis involves cumbersome sample processing and is costly; and weak anion exchange chromatography offers unsatisfactory separation results. Other existing methods for analyzing supercoiled plasmid DNA only separate the open circular structure of the plasmid, failing to effectively separate other DNA configurations (linear DNA, polynucleotide DNA, etc.). Furthermore, the molecular weight of the plasmid also poses challenges to the analytical methods used. Currently, for larger plasmids (above 10 kb), conventional electrophoresis methods are inadequate in separating circular and linear impurities, and HPLC methods are rarely reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights, and its application. This method ensures complete separation of supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA, and multiplying plasmid DNA from plasmid samples of various molecular weights. The method features high resolution and high sensitivity, and has significant application value in the field of nucleic acid drug analysis.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights, the method comprising:

[0008] Linearized plasmid samples and untreated plasmid samples for HPLC detection were prepared; HPLC detection was performed using an anion exchange column; chromatograms of supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA, and multiplying plasmid DNA in linearized and untreated plasmid samples of various molecular weights were collected; the mobile phase for HPLC detection included: mobile phase A was an aqueous solution containing tris(hydroxymethyl)aminomethane (Tris), and mobile phase B was an aqueous solution containing tris(hydroxymethyl)aminomethane and sodium chloride (NaCl); the elution program for HPLC detection included:

[0009] Time-phase A Mobile phase B

[0010] 0 minutes 100% 0%

[0011] 2 minutes 100%-P%P%

[0012] 24+T minutes 100%-Q%Q%

[0013] 25+T+U minutes 100% 0%

[0014] 35+T+U minutes 100% 0%;

[0015] The range of T values ​​is: 0 ≤ T ≤ 25; the range of U values ​​is: -5 ≤ U ≤ 10; the range of P values ​​is: 20 ≤ P ≤ 65; and the range of Q values ​​is: 10 ≤ Q ≤ 87.

[0016] "0≤T≤25", for example, it can be 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, etc.

[0017] "-5≤U≤10", for example, it can be -5, -7, -9, 0, 1, 3, 5, 7, 9 or 10, etc.

[0018] "20≤P≤65", for example, it can be 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65, etc.

[0019] "10≤Q≤87", for example, it can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 87, etc.

[0020] Preferably, the mobile phase A is an aqueous solution containing 10-50 mM tris(hydroxymethyl)aminomethane, such as 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM, and the pH is 7.40-7.60, such as 7.40, 7.50 or 7.60.

[0021] Preferably, the mobile phase A is an aqueous solution containing 20-25 mM tris(hydroxymethyl)aminomethane, for example, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM or 25 mM, with a pH of 7.40-7.60.

[0022] Preferably, the mobile phase B is an aqueous solution containing 10-50 mM tris(hydroxymethyl)aminomethane (e.g., 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM, etc.) and 0.5-1.5 M sodium chloride (e.g., 0.5 M, 0.7 M, 0.9 M, 1.0 M, 1.3 M or 1.5 M, etc.), with a pH of 7.40-7.60.

[0023] Preferably, the mobile phase B is an aqueous solution containing 20-25 mM tris(hydroxymethyl)aminomethane (e.g., 20 mM, 21 mM, 22 mM, 23 mM, 24 mM or 25 mM, etc.) and 0.8-1.2 M sodium chloride (e.g., 0.8 M, 0.9 M, 1.0 M, 1.1 M or 1.2 M, etc.), with a pH of 7.40-7.60.

[0024] Preferably, the functional group of the anion exchange chromatography column packing is a quaternary ammonium group.

[0025] Preferably, the particle size of the filler is 3-10 μm, for example, it can be 3, 5, 7, 9 or 10 μm.

[0026] Preferably, the inner diameter of the anion exchange column is 4.6 mm.

[0027] Preferably, the column temperature of the anion exchange chromatographic column is set to 20-40℃, for example, it can be 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, etc.

[0028] Preferably, the flow rate of the mobile phase is 0.2-0.6 mL / min, for example, it can be 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min or 0.6 mL / min, etc.

[0029] In this invention, the flow rate of the mobile phase refers to the total flow rate of mobile phase A and mobile phase B.

[0030] Preferably, the detection wavelength in the HPLC detection is set to 250-270 nm, for example, it can be 250 nm, 260 nm, 265 nm or 270 nm, etc., preferably 260 nm.

[0031] Preferably, the size of the plasmid sample is 4.0-15.0 kb, for example, it can be 4 kb, 8 kb or 14.5 kb, etc.

[0032] Preferably, the linearized plasmid sample is prepared by a method comprising the following steps: digesting an untreated plasmid sample, ultrapure water, restriction endonuclease and enzyme buffer in a water bath at 30-50℃ for 1-2 h, for example, at 30℃, 35℃, 40℃, 45℃ or 50℃, for example, for 1 h, 1.5 h or 2 h, and then inactivating the plasmid sample in a water bath at 60-90℃ to prepare a linearized sample, for example, at 60℃, 70℃, 80℃ or 90℃.

[0033] In this invention, the enzyme buffer may be, for example, rCutSmart buffer.

[0034] Preferably, the linearized plasmid sample and the untreated plasmid sample are diluted and mixed at a mass ratio of 1:(8-10).

[0035] Preferably, the untreated plasmid sample is selected from any one of supercoiled plasmid DNA, open circular plasmid DNA, or multiply plasmid DNA.

[0036] Preferably, the diluent used in the dilution mixture is an aqueous solution containing 5-50 mM tris(hydroxymethyl)aminomethane (e.g., 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM, etc.) and 0.5-5 mM disodium ethylenediaminetetraacetate, with a pH value of 7.90-8.10, such as 7.90, 8.00, or 8.10, etc.

[0037] Preferably, the diluent is an aqueous solution containing 10 mM tris(hydroxymethyl)aminomethane and 1 mM disodium ethylenediaminetetraacetate, with a pH of 7.90-8.10.

[0038] Preferably, the restriction endonuclease is used to cleave the restriction sites on the plasmid.

[0039] In this invention, the restriction endonuclease may be, for example, a common restriction site on plasmids such as EcoRV-HF and / or BspQI.

[0040] Preferably, the linearized plasmid size is 4.0-15.0 kb, for example, it can be 4.0 kb, 8.0 kb or 14.5 kb.

[0041] Preferably, the final concentration of the untreated plasmid sample in the mixture is 50-400 μg / μL, for example, it can be 50 μg / μL, 100 μg / μL, 150 μg / μL, 200 μg / μL, 250 μg / μL, 300 μg / μL, 350 μg / μL or 400 μg / μL.

[0042] Preferably, the final concentration of the restriction endonuclease in the mixture is 1-10 IU / μL, for example, it can be 1 IU / μL, 3 IU / μL, 5 IU / μL, 7 IU / μL, 9 IU / μL or 10 IU / μL, etc.

[0043] Preferably, the water bath enzymatic digestion temperature is 35-37℃, for example, it can be 35℃, 36℃ or 37℃.

[0044] Preferably, the water bath inactivation temperature is 75-80℃, for example, it can be 75℃, 76℃, 77℃, 78℃, 79℃ or 80℃, etc.

[0045] Secondly, the present invention provides the application of the method described in the first aspect for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights in nucleic acid analysis.

[0046] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights in this invention can effectively separate supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA, and aggregate plasmid DNA from plasmids of different sizes. Compared with electrophoresis, the method has higher detection accuracy and has important application value. Attached Figure Description

[0049] Figure 1The image shows a chromatogram of a 5 kb plasmid sample, where 1-4 represent open circular plasmid DNA, linear plasmid DNA, supercoiled plasmid DNA, and multiply DNA, respectively.

[0050] Figure 2 The image shows the chromatogram of an 8 kb plasmid sample. In the image, 1-4 represent open circular plasmid DNA, linear plasmid DNA, supercoiled plasmid DNA, and multiply DNA, respectively.

[0051] Figure 3 The image shows a chromatogram of a 14.5 kb plasmid sample. In the image, 1-4 represent open circular plasmid DNA, supercoiled plasmid DNA, linear plasmid DNA, and multiply DNA, respectively. Detailed Implementation

[0052] This invention provides a liquid chromatography method that ensures complete separation of supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA, and multiply plasmid DNA from plasmid samples of various molecular weights, comprising the following steps:

[0053] (1) Preparation of linearized and untreated plasmid samples for HPLC detection;

[0054] (2) Detection was performed using HPLC.

[0055] The detection methods used include:

[0056] (a) Mobile phase A is an aqueous solution containing 10-50 mM tris(hydroxymethyl)aminomethane, with a pH of 7.40-7.60. Mobile phase B is an aqueous solution containing 10-50 mM tris(hydroxymethyl)aminomethane and 0.5-1.5 M sodium chloride, with a pH of 7.40-7.60;

[0057] (b) Chromatographic separation was performed using an anion exchange column, with the following separation gradient program:

[0058] Time-phase A Mobile phase B

[0059] 0 minutes 100% 0%

[0060] 2 minutes 100%-P%P%

[0061] 24+T minutes 100%-Q%Q%

[0062] 25+T+U minutes 100% 0%

[0063] 35+T+U minutes 100% 0%;

[0064] The range of T values ​​is: 0 ≤ T ≤ 25; the range of U values ​​is: -5 ≤ U ≤ 10; the range of P values ​​is: 20 ≤ P ≤ 50; and the range of Q values ​​is: 10 ≤ Q ≤ 50.

[0065] (c) Chromatograms of supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA and multiply plasmid DNA in linearized-untreated plasmid samples of various molecular weights.

[0066] In some implementations, the elution procedure is as follows:

[0067] Time-phase A Mobile phase B

[0068] 0 minutes 100% 0%

[0069] 2 minutes 35% 65%

[0070] 24+ minutes 13% 87%

[0071] 25+T+U minutes 100% 0%

[0072] 35+T+U minutes 100% 0%;

[0073] The range of T values ​​is: 0 ≤ T ≤ 25; the range of U values ​​is: -5 ≤ U ≤ 10.

[0074] In some implementations, the elution procedure is as follows:

[0075] Time-phase A Mobile phase B

[0076] 0 minutes 100% 0%

[0077] 2 minutes 100%-P%P%

[0078] 24 minutes 100%-Q%Q%

[0079] 25 minutes 100% 0%

[0080] 35 minutes 100% 0%;

[0081] Among them, 20≤P≤65, and the range of Q values ​​is: 10≤Q≤87.

[0082] In some implementations, the detection using HPLC methods includes:

[0083] Mobile phase: Mobile phase A is an aqueous solution containing 10-50 mM tris(hydroxymethyl)aminomethane, with a pH of 7.40-7.60;

[0084] Mobile phase B is an aqueous solution containing 10-50 mM tris(hydroxymethyl)aminomethane and 0.5-1.5 M sodium chloride, with a pH of 7.40-7.60.

[0085] The elution procedure is as follows:

[0086] Time-phase A Mobile phase B

[0087] 0 minutes 100% 0%

[0088] 2 minutes 35% 65%

[0089] 24 minutes 13% 87%

[0090] 25 minutes 100% 0%

[0091] 35 minutes 100% 0%.

[0092] In one embodiment, the column temperature of the anion exchange column is set to 20°C.

[0093] In one embodiment, the column temperature of the anion exchange chromatography column is set to 25°C.

[0094] In one embodiment, the column temperature of the anion exchange column is set to 30°C.

[0095] In one embodiment, the column temperature of the anion exchange column is set to 35°C.

[0096] In one embodiment, the column temperature of the anion exchange column is set to 40°C.

[0097] In one embodiment, the flow rate of the mobile phase is 0.2 mL / min.

[0098] In one embodiment, the flow rate of the mobile phase is 0.3 mL / min.

[0099] In one embodiment, the flow rate of the mobile phase is 0.4 mL / min.

[0100] In one embodiment, the flow rate of the mobile phase is 0.5 mL / min.

[0101] In one embodiment, the flow rate of the mobile phase is 0.6 mL / min.

[0102] In one implementation, the HPLC instrument is an Agilent 1260 Infinity high-performance liquid chromatograph.

[0103] In one embodiment, the HPLC column is a BioCore SAX (5 μm, 4.6 × 250 mm) quaternary ammonium column.

[0104] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0105] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0106] Example 1: Chromatographic separation of 5 Kb plasmid samples

[0107] (1) Instruments and chromatographic conditions

[0108] Ultra-high performance liquid chromatograph: Agilent 1260 Infinity high performance liquid chromatograph, column: BioCore SAX (5 μm, 4.6 × 250 mm) column, detection wavelength: 260 nm, column temperature set to 30℃.

[0109] Prepare a 20 mM aqueous solution of tris(hydroxymethyl)aminomethane, adjust the pH to 7.50 with hydrochloric acid, and use this as mobile phase A. Prepare a mixture of 20 mM tris(hydroxymethyl)aminomethane and 1 M sodium chloride, adjust the pH to 7.50 with hydrochloric acid, and use this as mobile phase B. Set the flow rate to 0.5 mL / min, and the elution program is as follows:

[0110] Time-phase A Mobile phase B

[0111] 0 min 100% 0%

[0112] 2 min 35% 65%

[0113] 24 min 13% 87%

[0114] 25 min 100% 0%

[0115] 35 min 100% 0%.

[0116] (2) Experimental steps

[0117] Preparation of diluent: Prepare an aqueous solution of 10 mM tris(hydroxymethyl)aminomethane and 1 mM disodium ethylenediaminetetraacetate, and adjust the pH to 8.0 with hydrochloric acid.

[0118] Linearized sample preparation: Take 2 μL of restriction endonuclease BspQI, 10 μL of rCutSmart buffer, and a number of untreated 5 kb plasmid samples, and finally prepare a 100 μL mixture with ultrapure water. The content of untreated plasmid in the mixture is 4 μg. Place the mixture in a water bath at 37℃ for 2 h, and then inactivate it at 80℃ for 20 min to obtain the linearized sample.

[0119] Linearization + Untreated Sample Preparation: The linear + untreated sample is a mixture of linear plasmid DNA content: (supercoiled plasmid DNA content + open circular plasmid DNA content + multiply plasmid DNA content) = 1:9, with diluent as the solvent.

[0120] Take 20 μL of each of the above analytical solutions, inject it into the liquid chromatograph, and record the chromatogram. (See attached...) Figure 1 It can be seen that this method can completely separate supercoiled plasmid DNA, linear plasmid DNA, open circular plasmid DNA, and multiply plasmid DNA. The content of supercoiled plasmid DNA is its peak area percentage. The peak time, peak area percentage, and resolution data of plasmid DNA of each topological structure are shown in Table 1 below. Among them, the content of supercoiled plasmid DNA is 78.287%.

[0121] Table 1

[0122]

[0123] As shown in Table 1, 1-4 represent open circular plasmid DNA, linear plasmid DNA, supercoiled plasmid DNA, and multiplying DNA, respectively. Among them, the content of supercoiled plasmid DNA is 78.287%.

[0124] Example 2: Chromatographic separation of 8 kb plasmid samples

[0125] (1) Instruments and chromatographic conditions

[0126] Ultra-high performance liquid chromatograph: Agilent 1260 Infinity high performance liquid chromatograph, column: BioCore SAX (5 μm, 4.6 × 250 mm) column, detection wavelength: 260 nm, column temperature set to 30℃.

[0127] Prepare a 20 mM aqueous solution of tris(hydroxymethyl)aminomethane, adjust the pH to 7.50 with hydrochloric acid, and use this as mobile phase A. Prepare a mixture of 20 mM tris(hydroxymethyl)aminomethane and 1 M sodium chloride, adjust the pH to 7.50 with hydrochloric acid, and use this as mobile phase B. Set the flow rate to 0.5 mL / min, and the elution program is as follows:

[0128] Time-phase A Mobile phase B

[0129] 0 min 100% 0%

[0130] 2 min 35% 65%

[0131] 24 min 13% 87%

[0132] 25 min 100% 0%

[0133] 35 min 100% 0%.

[0134] (2) Experimental procedures

[0135] Preparation of diluent: Prepare an aqueous solution of 10 mM tris(hydroxymethyl)aminomethane and 1 mM disodium ethylenediaminetetraacetate and adjust the pH to 8.0 with hydrochloric acid.

[0136] Linearized sample preparation: Take 2 μL of restriction endonuclease EcoRV-HF, 10 μL of rCutSmart buffer, and a number of untreated 8 Kb plasmid samples, and finally prepare a 100 μL mixture with ultrapure water. The content of untreated plasmid in the mixture is 4 μg. Place the mixture in a water bath at 37℃ for 2 h, and then inactivate it at 80℃ for 20 min to obtain the linearized sample.

[0137] Linearization + Untreated Sample Preparation: The linear + untreated sample is a mixture of linear plasmid DNA content: (supercoiled plasmid DNA content + open circular plasmid DNA content + multiply plasmid DNA content) = 1:9, with diluent as the solvent.

[0138] Take 20 μL of each of the above analytical solutions, inject it into the liquid chromatograph, and record the chromatogram. Figure 2 As can be seen, this method can completely separate supercoiled plasmid DNA, linear plasmid DNA, open circular plasmid DNA, and multiply plasmid DNA. The supercoiled plasmid DNA content is its peak area percentage. The elution time, peak area percentage, and resolution data of plasmid DNA with different topologies are shown in Table 2 below.

[0139] Table 2

[0140]

[0141] Table 2 shows that 1-4 represent open circular plasmid DNA, linear plasmid DNA, supercoiled plasmid DNA, and multiplying DNA, respectively. Among these, supercoiled plasmid DNA accounted for 83.83%.

[0142] Example 3: Chromatographic separation of a 14.5 kb plasmid sample

[0143] (1) Instruments and chromatographic conditions

[0144] Ultra-high performance liquid chromatograph: Agilent 1260 Infinity high performance liquid chromatograph, with a BioCore SAX (5 μm, 4.6 × 250 mm) column, a detection wavelength of 260 nm, and a column temperature of 30 °C.

[0145] Prepare a 20 mM aqueous solution of tris(hydroxymethyl)aminomethane, adjust the pH to 7.50 with hydrochloric acid, and use this as mobile phase A. Prepare a mixture of 20 mM tris(hydroxymethyl)aminomethane and 1 M sodium chloride, adjust the pH to 7.50 with hydrochloric acid, and use this as mobile phase B. Set the flow rate to 0.5 mL / min, and the elution program is as follows:

[0146] Time-phase A Mobile phase B

[0147] 0 min 100% 0%

[0148] 2 min 35% 65%

[0149] 24 min 13% 87%

[0150] 25 min 100% 0%

[0151] 35 min 100% 0%.

[0152] (2) Experimental steps

[0153] Preparation of diluent: Prepare an aqueous solution of 10 mM tris(hydroxymethyl)aminomethane and 1 mM disodium ethylenediaminetetraacetate and adjust the pH to 8.0 with hydrochloric acid.

[0154] Linearized sample preparation: Take 2 μL of restriction endonuclease EcoRV-HF, 10 μL of rCutSmart buffer, and several untreated 14.5 kb plasmid samples, and finally prepare a 100 μL mixture with ultrapure water. The content of untreated plasmid in the mixture is 4 μg. Place the mixture in a water bath at 37℃ for 2 h, and then inactivate it at 80℃ for 20 min to obtain the linearized sample.

[0155] Linearization + Untreated Sample Preparation: The linear + untreated sample is a mixture of linear plasmid DNA content: (supercoiled plasmid DNA content + open circular plasmid DNA content + multiply plasmid DNA content) = 1:9, with diluent as the solvent.

[0156] Take 20 μL of each of the above analytical solutions, inject it into the liquid chromatograph, and record the chromatogram. (See attached...) Figure 3 As can be seen, this method can completely separate supercoiled plasmid DNA, linear plasmid DNA, open circular plasmid DNA, and multiplying plasmid DNA. The supercoiled plasmid DNA content is its peak area percentage. The elution time, peak area percentage, and resolution data of plasmid DNA with different topologies are shown in Table 3 below.

[0157] Table 3

[0158]

[0159] Table 3 shows that 1-4 represent open circular plasmid DNA, supercoiled plasmid DNA, linear plasmid DNA, and multiplying DNA, respectively. The content of supercoiled plasmid DNA is 82.858%.

[0160] In summary, this invention provides a liquid chromatography method that can guarantee the complete separation of supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA, and multiply plasmid DNA from plasmid samples of various molecular weights. The method has high resolution and high sensitivity, and has important application value in the field of nucleic acid drug analysis technology.

[0161] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for detecting the purity of supercoiled DNA in a plasmid sample of different molecular weights, characterized in that, The method includes: Linearized plasmid samples and untreated plasmid samples were prepared for HPLC detection; HPLC detection was performed using an anion exchange column; chromatograms of supercoiled plasmid DNA, open circular plasmid DNA, linear plasmid DNA, and multiplying plasmid DNA were collected from linearized plasmid samples and untreated plasmid samples of various molecular weights; the size of the plasmid samples was 5-15.0 kb. The mobile phase for HPLC detection comprises: mobile phase A, an aqueous solution containing 20-25 mM tris(hydroxymethyl)aminomethane, adjusted to pH 7.40-7.60 with hydrochloric acid; mobile phase B, an aqueous solution containing 20-25 mM tris(hydroxymethyl)aminomethane and 0.8-1.2 M sodium chloride, adjusted to pH 7.40-7.60 with hydrochloric acid; the flow rate of the mobile phase is 0.2-0.6 mL / min; and the detection wavelength in the HPLC detection is set to 250-270 nm. The elution procedure for HPLC detection includes: Time-phase A Mobile phase B 0 min 100% 0% 2 min 35% 65% 24 min 13% 87% 25 min 100% 0% 35 min 100% 0%; The functional group of the anion exchange chromatography column packing is quaternary ammonium group; the particle size of the packing is 5 μm; the inner diameter of the anion exchange chromatography column is 4.6 mm and the column length is 250 mm; the column temperature of the anion exchange chromatography column is set to 20-40℃.

2. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 1, characterized in that, The detection wavelength in the HPLC detection was set to 260 nm.

3. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 1, characterized in that, The linearized plasmid sample was prepared by a method including the following steps: a mixture of untreated plasmid sample, ultrapure water, restriction endonuclease and enzyme buffer was digested in a water bath at 30-50℃ for 1-2 h, and then inactivated in a water bath at 60-90℃ to prepare a linearized plasmid sample.

4. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 1, characterized in that, The linearized plasmid sample and the untreated plasmid sample were diluted and mixed at a mass ratio of 1:(8-10).

5. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 4, characterized in that, The diluent used in the dilution mixture is an aqueous solution containing 5-50 mM tris(hydroxymethyl)aminomethane and 0.5-5 mM disodium ethylenediaminetetraacetate, with a pH of 7.90-8.

10.

6. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 5, characterized in that, The diluent is an aqueous solution containing 10 mM tris(hydroxymethyl)aminomethane and 1 mM disodium ethylenediaminetetraacetate, with a pH of 7.90-8.

10.

7. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 3, characterized in that, The restriction endonuclease is used to cleave the restriction sites on the plasmid.

8. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 3, characterized in that, The final concentration of the untreated plasmid sample in the mixture is 50-400 μg / μL.

9. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 3, characterized in that, The final concentration of restriction endonuclease in the mixture is 1-10 IU / μL.

10. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 3, characterized in that, The water bath enzymatic digestion temperature is 35-37℃.

11. The method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to claim 3, characterized in that, The water bath inactivation temperature is 75-80℃.

12. The application of the method for detecting the purity of supercoiled DNA in plasmid samples of different molecular weights according to any one of claims 1-11 in nucleic acid analysis.

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