A linear DNA end integrity assay based on liquid chromatography-mass spectrometry
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI JIABEI BIOMEDICAL TECH CO LTD
- Filing Date
- 2023-11-09
- Publication Date
- 2026-06-09
Smart Images
Figure HDA0004539305330000011 
Figure HDA0004539305330000012 
Figure HDA0004539305330000013
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry (LC-MS). Background Technology
[0002] PCR products, linearization of circular plasmid DNA (template for mRNA synthesis in in vitro reverse transcription), and DNA fragments after enzymatic digestion or sonication can all introduce sticky ends into DNA fragments. To obtain blunt ends, these DNA fragments need end repair. The purpose of end repair is to restore damaged or incomplete DNA ends to 5' phosphorylated blunt ends. End-repair enzymes, such as T4 DNA polymerase, possess 5'→3' DNA polymerase activity and 3'→5' DNA exonuclease activity, and can be used to convert sticky ends generated after enzyme digestion or PCR reactions into blunt ends.
[0003] Whether the ends of linear DNA are padded (i.e., the integrity of the ends of linear DNA) can be determined by conventional detection methods such as acrylamide gel electrophoresis and capillary electrophoresis. However, due to their limitations in resolution and accuracy, they cannot obtain precise sequence information. These detection methods cannot meet the quality control requirements of biopharmaceuticals (high versatility; high accuracy, specificity and sensitivity). Summary of the Invention
[0004] To meet the quality control requirements of molecular biology and biopharmaceuticals, this invention employs liquid chromatography-mass spectrometry (LC-MS) to accurately characterize and relatively quantify the sequences at both ends of DNA based on the differences in the molecular weight of DNA fragments, providing a method for determining the end integrity of linear DNA based on LC-MS.
[0005] The specific technical solution of the present invention is as follows:
[0006] The present invention provides a method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry (LC-MS), characterized by the following steps: Step S1, selecting appropriate restriction enzyme sites at the 5' end and / or 3' end of the linear DNA, and selecting restriction endonucleases at the 5' end and / or 3' end according to the restriction enzyme sites; Step S2, digesting the linear DNA with restriction endonucleases to obtain a digested solution; Step S3, separating the digested 5' end DNA double-stranded fragments and / or the digested 3' end DNA double-stranded fragments from the digested solution using DNA length sorting magnetic beads, and collecting the supernatant to obtain the SQ1 sample; Step S4, precipitating the SQ1 sample with ethanol to replace the buffer, obtaining the sample to be tested; Step S5, performing LC-MS on the sample to be tested.
[0007] The linear DNA end integrity determination method based on liquid chromatography-mass spectrometry provided by this invention also has the following technical features, wherein the specific conditions for selecting restriction endonucleases in step S1 are as follows: the sequence of linear DNA is imported into nucleic acid analysis software, and a suitable restriction endonuclease site is selected so that the lengths of the 5' end DNA double-stranded fragment and the 3' end DNA double-stranded fragment after enzyme digestion are less than 100 bp.
[0008] The method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry (LC-MS) provided by this invention also has the following technical features: The specific process of enzyme digestion in step S2 is as follows: A restriction endonuclease is added to the linear DNA, followed by the addition of a buffer compatible with the restriction endonuclease and sterile, enzyme-free water. After mixing, the DNA is digested. When digestion of the 5' and 3' ends of the linear DNA is required, the digestion process is as follows: If the buffers compatible with the 5' and 3' end restriction endonucleases are different, the 5' and 3' ends of the linear DNA are digested separately. Specifically, the digestion process is as follows: A 5' end restriction endonuclease is added to the linear DNA, followed by the addition of a buffer compatible with the 5' end restriction endonuclease and sterile, enzyme-free water. After mixing, the DNA is digested to obtain a first digested solution. This first digested solution contains the digested 5' end DNA double-stranded fragment and the remaining fragments larger than 1... A 0.00 bp DNA double-stranded fragment; add a 3' restriction endonuclease to another batch of linear DNA, add the buffer compatible with the 3' restriction endonuclease and sterile enzyme-free water, mix well, and then perform enzymatic digestion to obtain a second digested solution. This second digested solution contains the digested 3' end DNA double-stranded fragment and other DNA double-stranded fragments larger than 100 bp; if the buffers compatible with the 5' and 3' restriction endonucleases are the same, the 5' and 3' ends of the linear DNA can be digested simultaneously. The specific digestion process is as follows: add the 5' and 3' restriction endonucleases to the linear DNA simultaneously, add the buffer compatible with the restriction endonuclease and sterile enzyme-free water, mix well, and then perform enzymatic digestion to obtain a digested solution. This digested solution contains both the digested 5' end DNA double-stranded fragment and the digested 3' end DNA double-stranded fragment.
[0009] The linear DNA end integrity determination method based on liquid chromatography-mass spectrometry provided by this invention also has the following technical features: in step S3, the DNA length sorting magnetic beads are magnetic beads that can adsorb DNA double-stranded fragments with a length greater than 100 bp; the specific purification process is as follows: after the DNA length sorting magnetic bead solution is mixed evenly, it is added to the enzyme digestion solution, and after being thoroughly mixed, it is incubated at room temperature for 5-15 min to allow DNA double-stranded fragments with a length greater than 100 bp to adsorb onto the magnetic beads. The incubated solution is placed on a magnetic rack, and after the solution is clarified, the supernatant is aspirated to obtain the SQ1 sample.
[0010] The method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry (LC-MS) provided by this invention also has the following technical features: the specific process of step S4, ethanol precipitation and buffer replacement, is as follows: add anhydrous ethanol and 4.5-5.5 mol / L ammonium acetate solution to the SQ1 sample, mix well, and react on ice for 20-40 min; after the reaction, centrifuge at 14000-16000 rpm for 10-20 min at 3-5℃, and discard the supernatant; add 70% ethanol solution pre-cooled at -22℃ to -18℃, mix well, centrifuge at 14000-16000 rpm for 10-20 min at 3-5℃, discard the supernatant, and repeat this step once; after air drying at room temperature, reconstitute with sterile enzyme-free water to obtain the sample to be tested.
[0011] The method for determining the end integrity of linear DNA based on LC-MS provided by this invention also has the following technical features: the LC-MS detection conditions for step S5 are as follows: capillary voltage of ESI: Negative is 2.0-3.0kV, sampling cone voltage is 35-45V, source temperature is 140-160℃, desolvation temperature is 440-460℃, and mass range is 400-5000m / z.
[0012] The role and effect of invention
[0013] This invention allows for the selection of specific restriction endonucleases based on linear DNA sequences to obtain 5' and / or 3' double-stranded DNA fragments shorter than 100 bp. It also enables the effective enrichment and purification of these fragments. Liquid chromatography-mass spectrometry (LC-MS) allows for precise sequence characterization and relative content analysis of the short fragments (5' and / or 3' ends) after LC-MS digestion. The linear DNA end integrity determination method provided by this invention offers advantages such as high versatility, accuracy, specificity, and sensitivity, enabling sequence analysis of linear DNA ends (including determining whether blunt ends are obtained after enzyme completion of sticky DNA ends). Attached Figure Description
[0014] Figure 1 This is a schematic diagram of the enzyme digestion of the linear DNA sequence in Example 1 of the present invention.
[0015] Figure 2 This is the total ion chromatogram (TIC) of the 5' end DNA double-stranded fragment in sample 1 of Example 1 of the present invention.
[0016] Figure 3 This is a magnified image (23000-27000 Da) of the deconvolution of the 5' end DNA double-stranded fragment in sample 1 of Example 1 of the present invention at 9.92 min.
[0017] Figure 4This is the total ion chromatogram (TIC) of the 3' end DNA double-stranded fragment in sample 2 of Example 1 of the present invention.
[0018] Figure 5 This is a magnified image (5500-6800 Da) of the deconvolution of the 3' end DNA double-stranded fragment in sample 2 of Example 1 of the present invention at 7.52 min.
[0019] Figure 6 This is a magnified image (6000-7300 Da) of the deconvolution of the 3' end DNA double-stranded fragment in sample 2 of Example 1 of the present invention at 8.22 min.
[0020] Figure 7 This is the total ion chromatogram (TIC) of the 5' end DNA double-stranded fragment in the sample to be tested in Example 2 of the present invention.
[0021] Figure 8 This is a magnified image (22000-27000 Da) of the deconvolutioned 5' end DNA double-stranded fragment in the sample to be tested in Example 2 of the present invention at 9.82 min. Detailed Implementation
[0022] The terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.
[0023] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.
[0024] The reagents used in the following examples were obtained through common commercial channels. Experimental procedures and conditions not specified are in accordance with conventional procedures and conditions in the art.
[0025] The specific embodiments of the present invention will be described below with reference to the examples and accompanying drawings.
[0026] <Example 1>
[0027] This embodiment provides a method for determining the end integrity of linear DNA using liquid chromatography-mass spectrometry (LC-MS), which measures the 5' and 3' ends of linear DNA. The method includes the following steps:
[0028] Step S1 involves selecting appropriate restriction enzyme sites at the 5' and 3' ends of the linear DNA, and then selecting restriction endonucleases at the 5' and 3' ends based on these sites. The specific process is as follows:
[0029] The linear DNA sequence was imported into Snap Gene analysis software. Appropriate restriction enzyme sites were selected at the 5' and 3' ends of the linear DNA, ensuring that the lengths of both the 5' and 3' double-stranded DNA fragments after digestion were less than 100 bp. The restriction endonucleases were specific and commercially available.
[0030] After analysis using Snap Gene software, the selected 5' restriction endonuclease was AseI (NEB, catalog number R0526S), and the selected 3' restriction endonuclease was BsaAI (NEB, catalog number R0531S).
[0031] A schematic diagram of linear DNA digestion using AseI and BsaAI restriction endonucleases is shown below. Figure 1 As shown, the sequences indicated by the arrows are named 5-F, 5-R, 3-F, and 3-R, respectively. Because the linear DNA sequence used in this embodiment needs to be kept confidential, a portion of the base sequence has been hidden. This hidden portion does not affect the implementation of the assay method in this embodiment, nor does it affect the applicability of the assay method to the determination of the end integrity of other linear DNA sequences.
[0032] Step S2 involves digesting the linear DNA with restriction endonucleases to obtain a digested solution. The specific process is as follows:
[0033] Because the buffers for AseI and BsaAI restriction endonucleases are different, the 5' and 3' ends of linear DNA need to be digested separately. The specific process is as follows:
[0034] 5' end restriction enzyme digestion:
[0035] Take 100 μg of linear DNA fragment, add 50 μl of AseI restriction endonuclease, add 50 μl of NEBuffer r3.1, add sterile enzyme-free water to 500 μl, vortex to mix, and digest at 37℃ for 3 h to obtain the first digestion solution. This first digestion solution contains the 5' end DNA double-stranded fragment after digestion and the remaining DNA double-stranded fragments larger than 100 bp.
[0036] 3' end restriction enzyme digestion:
[0037] Take another 100 μg of the same linear DNA fragment, add 100 μl of BsaAI restriction endonuclease, add 50 μl of CutSmart Buffer, add sterile enzyme-free water to 500 μl, vortex to mix, and digest at 37°C for 3 h to obtain the second digested solution. This second digested solution contains the 3' end DNA double-stranded fragment after digestion and the remaining DNA double-stranded fragments larger than 100 bp.
[0038] If the restriction endonucleases for the 5' and 3' ends are packaged with the same buffer, the 5' and 3' ends of linear DNA can be digested simultaneously.
[0039] Step S3 involves using DNA length sorting magnetic beads to separate the 5' and 3' double-stranded DNA fragments from the enzyme-digested solution, and collecting the supernatant to obtain sample SQ1. The specific process is as follows:
[0040] Remove the VAHTS DNA Clean Beads solution (Vazyme, catalog number N411) from 2-8℃ 30 minutes in advance and allow it to warm to room temperature. Invert or vortex to thoroughly mix the magnetic bead solution. Add 600 μl of the magnetic bead solution to the solution after the first enzyme digestion and gently pipette 10 times to mix thoroughly. Incubate at room temperature for 10 minutes to allow DNA double-stranded fragments longer than 100 bp to adsorb onto the magnetic beads. Place the incubated solution on a magnetic rack and wait for the solution to clarify (about 5 minutes). Carefully aspirate the supernatant to obtain the SQ1-1 sample (about 1 ml).
[0041] Remove the VAHTS DNA Clean Beads solution (Vazyme, catalog number N411) from 2-8°C 30 minutes in advance and allow it to warm to room temperature. Invert or vortex to thoroughly mix the magnetic bead solution. Add 600 μl of the magnetic bead solution to the solution after the second enzyme digestion and gently pipette 10 times to mix thoroughly. Incubate at room temperature for 10 minutes to allow DNA double-stranded fragments longer than 100 bp to adsorb onto the magnetic beads. Place the incubated solution on a magnetic rack and wait for the solution to clarify (about 5 minutes). Carefully aspirate the supernatant to obtain the SQ1-2 sample (about 1 ml).
[0042] Step S4: Ethanol precipitation and buffer replacement are applied to sample SQ1 to obtain the sample to be tested. The specific process for applying ethanol precipitation and buffer replacement to sample SQ1-1 is as follows:
[0043] Take 250 μl of SQ1-1 sample / tube (4 tubes in total), add 687.5 μl of anhydrous ethanol to each tube, add 5 μl of 5 mol / L ammonium acetate solution, vortex to mix, and react on ice for 30 min; after the reaction, centrifuge at 15000 rpm for 15 min at 4℃, and discard the supernatant; add 1 ml of 70% ethanol solution pre-cooled at -20℃, invert 10 times, centrifuge at 15000 rpm for 15 min at 4℃, discard the supernatant, and then repeat this step once; air dry at room temperature for 10 min, and reconstitute with 28 μl of sterile enzyme-free water to obtain sample 1 to be tested.
[0044] The process of ethanol precipitation and buffer replacement for sample SQ1-2 was the same as that for sample SQ1-1, resulting in sample 2 to be tested.
[0045] Step S5: Perform LC-MS analysis on the sample to be tested. The specific process is as follows:
[0046] 20 μl of each of the two test samples (sample 1 and sample 2) were transferred to the inner tubes of two vials for LC-MS analysis. The LC-MS detection conditions were as follows: capillary voltage of ESI: Negative was 2.5 kV, sampling cone voltage was 40 V, source temperature was 150 °C, desolvation temperature was 450 °C, and mass range was 400-5000 m / z.
[0047] Test results as follows Figure 2-6 As shown. Figure 2 This is the total ion chromatogram (TIC) of the 5' end DNA double-stranded fragment in sample 1 of Example 1 of the present invention. Figure 3 This is a magnified image (23000-27000 Da) of the deconvolution of the 5' end DNA double-stranded fragment in sample 1 of Example 1 of the present invention at 9.92 min. Figure 4 This is the total ion chromatogram (TIC) of the 3' end DNA double-stranded fragment in sample 2 of Example 1 of the present invention. Figure 5 This is a magnified image (5500-6800 Da) of the deconvolution of the 3' end DNA double-stranded fragment in sample 2 of Example 1 of the present invention at 7.52 min. Figure 6 This is a magnified image (6000-7300 Da) of the deconvolution of the 3' end DNA double-stranded fragment in sample 2 of Example 1 of the present invention at 8.22 min.
[0048] Depend on Figure 2-6 It can be seen that the measured molecular weights of the two single-stranded 5-F and 5-R segments of the 5' end fragment after enzyme digestion are consistent with the theoretical sequence (e.g., Figure 2-3 As shown), the measured molecular weights of the two single-stranded 3-F and 3-R segments of the 3' end fragment after enzyme digestion are consistent with the theoretical sequence (e.g., Figure 4-6 (As shown).
[0049] <Example 2>
[0050] This embodiment provides a method for determining the 5' end integrity of linear DNA using liquid chromatography-mass spectrometry (LC-MS), which measures the 5' end of linear DNA. The method includes the following steps:
[0051] Step S1 involves selecting a suitable restriction enzyme site at the 5' end of the linear DNA, and then selecting a restriction endonuclease at the 5' end based on the restriction site. The specific process is as follows:
[0052] The sequence of linear DNA (consistent with that in Example 1) was imported into the Snap Gene analysis software. A suitable restriction enzyme site was selected at the 5' end of the linear DNA, ensuring that the length of the 5' end double-stranded DNA fragment after enzyme digestion was less than 100 bp. The restriction endonuclease must be specific and commercially available.
[0053] The 5' restriction endonuclease selected after analysis using Snap Gene software was AseI restriction endonuclease (NEB, catalog number R0526S).
[0054] Schematic diagram of linear DNA digestion using AseI restriction endonuclease and Example 1. Figure 1 Except for deleting the portion digested by the BsaAI restriction endonuclease, the rest of the digestion process is the same.
[0055] Step S2 involves digesting the linear DNA with restriction endonucleases to obtain a digested solution. The specific process is as follows:
[0056] Take 100 μg of linear DNA fragment, add 50 μl of AseI restriction endonuclease, add 50 μl of NEBuffer r3.1, add sterile enzyme-free water to 500 μl, vortex to mix, and digest at 37℃ for 3 h to obtain the digested solution. The digested solution contains the 5' end DNA double-stranded fragment after digestion and the remaining DNA double-stranded fragments larger than 100 bp.
[0057] Step S3: Using DNA length sorting magnetic beads, the 5' end of the double-stranded DNA fragment after enzyme digestion is separated from the digested solution, and the supernatant is collected to obtain the SQ1 sample. The specific process is as follows:
[0058] Remove the VAHTS DNA Clean Beads solution (Vazyme, catalog number N411) from 2-8℃ 30 minutes in advance and allow it to warm to room temperature. Invert or vortex to thoroughly mix the magnetic bead solution. Add 600 μl of the magnetic bead solution to the digested solution and gently pipette 10 times to mix thoroughly. Incubate at room temperature for 10 minutes to allow DNA double-stranded fragments longer than 100 bp to adsorb onto the magnetic beads. Place the incubated solution on a magnetic rack and wait for the solution to clarify (about 5 minutes). Carefully aspirate the supernatant to obtain the SQ1 sample (about 1 ml).
[0059] Step S4: Ethanol precipitation is performed on sample SQ1 to replace the buffer, yielding the sample to be tested. The specific process is as follows:
[0060] Take 250 μl of SQ1 sample per tube (4 tubes in total), add 687.5 μl of anhydrous ethanol to each tube, add 5 μl of 5 mol / L ammonium acetate solution, vortex to mix, and react on ice for 30 min. After the reaction, centrifuge at 15000 rpm for 15 min at 4 °C and discard the supernatant. Add 1 ml of 70% ethanol solution pre-cooled at -20 °C, invert 10 times, centrifuge at 15000 rpm for 15 min at 4 °C, discard the supernatant, and then repeat this step once. Air dry at room temperature for 10 min, and reconstitute with 28 μl of sterile enzyme-free water to obtain the sample to be tested.
[0061] Step S5: Perform LC-MS analysis on the sample to be tested. The specific process is as follows:
[0062] Take 20 μl of the sample to be tested and transfer it to the inner tube of the vial for LC-MS analysis. The LC-MS detection conditions are as follows: capillary voltage of ESI: Negative is 2.5 kV, sampling cone voltage is 40 V, source temperature is 150 °C, desolvation temperature is 450 °C, and mass range is 400-5000 m / z.
[0063] Test results as follows Figure 7-8 As shown. Figure 7 This is the total ion chromatogram (TIC) of the 5' end DNA double-stranded fragment in the sample to be tested in Example 2 of the present invention. Figure 8 This is a magnified image (22000-27000 Da) of the deconvolutioned 5' end DNA double-stranded fragment in the sample to be tested in Example 2 of the present invention at 9.82 min.
[0064] Depend on Figure 7-8 It was found that the 5' end fragments after enzyme digestion showed that the 5'-F single strand had one more base than the theoretical sequence, and also two or three fewer bases; the 5'-R single strand was also not completed, showing reductions of three, one, and five bases compared to the theoretical sequence. In other words, the 5'-F strand had sequences with increased or decreased bases, while no completed sequence was detected in the 5'-R strand, meaning that no blunt-ended DNA fragments were obtained after polymerase treatment.
[0065] The detection results of Examples 1 and 2 show that the linear DNA end integrity determination method provided by the present invention can realize the analysis of the end sequence of linear DNA and determine whether the DNA has obtained blunt ends after enzyme treatment.
[0066] The above is a detailed description of the embodiments, which is intended to enable those skilled in the art to correctly understand and use the present invention. Any improvements or modifications to technical solutions obtained by those skilled in the art based on the present invention and on the existing technology, without innovative effort but only through analysis, analogy, or limited enumeration, should be within the scope of protection defined by the claims.
Claims
1. A method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry (LC-MS), characterized in that, Includes the following steps: Step S1: Select appropriate restriction enzyme sites at the 5' end and / or 3' end of the linear DNA, and select restriction endonucleases at the 5' end and / or 3' end according to the restriction enzyme sites. Step S2: The linear DNA is digested with the restriction endonuclease to obtain a digested solution. Step S3: Use DNA length sorting magnetic beads to separate the 5' end DNA double-stranded fragment and / or the 3' end DNA double-stranded fragment after enzyme digestion from the enzyme digestion solution, and collect the supernatant to obtain the SQ1 sample; Step S4: Perform ethanol precipitation to replace the buffer in the SQ1 sample to obtain the sample to be tested; Step S5: Perform LC-MS on the sample to be tested. The specific process of enzyme digestion in step S2 is as follows: The restriction endonuclease was added to the linear DNA, followed by the addition of the buffer containing the restriction endonuclease and sterile, enzyme-free water. After mixing thoroughly, the DNA was digested. When it is necessary to digest the 5' and 3' ends of linear DNA, the digestion process is as follows: If the buffers used for the restriction endonucleases at the 5' and 3' ends are different, the 5' and 3' ends of the linear DNA are digested separately. The specific digestion process is as follows: Add the 5' restriction endonuclease to the linear DNA, add the buffer used for the 5' restriction endonuclease and sterile enzyme-free water, mix well, and then digest to obtain the first digested solution. This first digested solution contains the digested 5' end DNA double-stranded fragment and other DNA double-stranded fragments longer than 100 bp. Add the 3' restriction endonuclease to another batch of the linear DNA, add the buffer used for the 3' restriction endonuclease and sterile enzyme-free water, mix well, and then digest to obtain the second digested solution. This second digested solution contains the digested 3' end DNA double-stranded fragment and other DNA double-stranded fragments longer than 100 bp. If the buffers for the restriction endonucleases at the 5' and 3' ends are the same, the 5' and 3' ends of the linear DNA can be digested simultaneously. The specific digestion process is as follows: add the restriction endonucleases for the 5' and 3' ends to the linear DNA at the same time, add the buffers for the restriction endonucleases and sterile enzyme-free water, mix well, and then perform digestion to obtain a digested solution. This digested solution contains both the digested 5' end DNA double-stranded fragment and the digested 3' end DNA double-stranded fragment. The LC-MS detection conditions for the on-machine testing described in step S5 are as follows: ESI: Negative has a capillary voltage of 2.0-3.0kV, a sampling cone voltage of 35-45V, a source temperature of 140-160℃, a desolventizing temperature of 440-460℃, and a mass range of 400-5000m / z.
2. The method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, in, The specific conditions for the restriction endonuclease mentioned in step S1 are as follows: The sequence of the linear DNA was imported into nucleic acid analysis software, and appropriate restriction endonuclease sites were selected to ensure that the lengths of the 5' and 3' DNA double-stranded fragments after restriction enzyme digestion were less than 100 bp.
3. The method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, in, The DNA length sorting magnetic beads mentioned in step S3 are magnetic beads that can adsorb DNA double-stranded fragments with a length greater than 100bp; The specific process of step S3 is as follows: After mixing the DNA length sorting magnetic bead solution, add it to the enzyme digestion solution. After mixing thoroughly, incubate at room temperature for 5-15 minutes to allow DNA double-stranded fragments longer than 100 bp to adsorb onto the magnetic beads. Place the incubated solution on a magnetic rack and wait for the solution to clarify. Then, aspirate the supernatant to obtain the SQ1 sample.
4. The method for determining the end integrity of linear DNA based on liquid chromatography-mass spectrometry according to claim 1, characterized in that, in, The specific process of ethanol precipitation and buffer replacement in step S4 is as follows: Add anhydrous ethanol and 4.5-5.5 mol / L ammonium acetate solution to the SQ1 sample, mix well, and react on ice for 20-40 min. After the reaction, centrifuge at 14000-16000 rpm for 10-20 min at 3-5℃ and discard the supernatant. Add 70% ethanol solution pre-cooled at -22℃ to -18℃, mix well, and centrifuge at 14000-16000 rpm for 10-20 min at 3-5℃ and discard the supernatant. Repeat this step once. After air-drying at room temperature, reconstitute with sterile enzyme-free water to obtain the sample to be tested.