Anion exchange high performance liquid chromatography mobile phase system, method and application for separating and purifying long single-stranded DNA

Abstract

This invention discloses a mobile phase system, purification method, and application for the separation and purification of long single-stranded DNA by anion exchange high-performance liquid chromatography (HPLC). A complete mobile phase system is constructed based on 20 mM NaOH and compounded with 1.5 M NaClO4. The mobile phase system consists of an equilibration buffer, binding buffer, washing buffer, elution buffer, regeneration buffer 1, and regeneration buffer 2. This system can stably separate long single-stranded DNA ranging from hundreds to thousands of nt. Compared with the NaCl system, the liquid phase recovery rate is increased by more than 15%, and the product purity is consistently ≥98%. The entire process is highly automated and reproducible, supporting gram-scale production. It solves the technical problem of traditional purification methods being difficult to industrialize and can be widely applied to the standardized preparation of long single-stranded DNA raw materials in fields such as gene therapy, synthetic biology, and DNA information storage.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and separation science and technology, and more specifically to a mobile phase system, method and application for the separation and purification of long single-stranded DNA by anion exchange high-performance liquid chromatography. Background Technology

[0002] Long single-stranded DNA (base length > 100 nt) is a core raw material for gene therapy, synthetic biology, epigenetic regulation, and DNA information storage. It can be prepared through chemical synthesis, reverse transcription, enzymatic digestion, asymmetric PCR, RCA, and phage biosynthesis. However, regardless of the preparation route, the crude product obtained is contaminated with impurities such as short oligonucleotides, primer dimers, incompletely cleaved long heterochain nucleic acids, and residual RNA. It must be purified to meet the requirements of downstream experiments and industrial applications.

[0003] Currently, the mainstream long single-stranded DNA purification methods in the industry each have significant shortcomings: polyacrylamide gel and denaturing agarose gel excision purification have high resolution, but the operation process is cumbersome and time-consuming, with a purification recovery rate of only about 60%, making it difficult to automate batch processing; the purification process introduces nucleic acid dyes, gel polymers and other difficult-to-remove impurities, and can only achieve microgram-level small-scale preparation, which is completely unsuitable for large-scale production; magnetic bead purification relies on nucleic acid-specific modification or complementary pairing capture, which has poor versatility and cannot universally separate long single-stranded DNA of any sequence and length; ordinary atmospheric pressure chromatography columns have insufficient resolution, can only complete simple desalting, and the recovery efficiency of long single-stranded DNA is unstable, and the separation effect is difficult to reproduce.

[0004] Anion exchange high-performance liquid chromatography (HPLC) achieves separation based on the difference in negative charge density of the nucleic acid phosphate backbone. It is naturally adapted for the fractionation of single-stranded DNA of different lengths and offers advantages such as automation, high reproducibility, scalability, and high product purity, making it a preferred route for nucleic acid purification. However, existing anion exchange chromatography techniques have significant limitations:

[0005] Existing commercial and patented NaCl-type anion exchange processes can only stably separate single-stranded DNA with a base length of less than 300 nt; Chinese invention patent CN121591819A can only purify chemically synthesized long single-stranded DNA of 250~300 nt, and cannot effectively separate ultra-long single-stranded DNA of hundreds to thousands of nt.

[0006] Existing literature discloses anion exchange separation schemes that are only developed for 1 kb~15 kb double-stranded DNA and plasmid DNA. However, double-stranded DNA and single-stranded DNA have very different molecular structures, spatial secondary structures, and surface charge distributions. Elution buffers and gradient processes for double-stranded DNA cannot be directly applied to the separation of long single-stranded DNA. Long single-stranded DNA is prone to forming secondary structures such as hairpins and stem loops, which interfere with anion exchange adsorption and elution. Conventional NaCl buffer systems cannot eliminate this interference, resulting in peak tailing of the target product, inability to baseline separate long and short fragments, and a significant decrease in recovery rate.

[0007] Traditional gel purification has limited recovery rates and cannot be scaled up; magnetic bead purification lacks versatility; existing NaCl-type anion exchange chromatography is only suitable for short fragments, cannot eliminate interference from long single-strand secondary structures, and lacks a complete set of standardized processes, making it difficult to achieve stable, efficient, and scalable automated purification of single-stranded DNA of hundreds to thousands of base pairs.

[0008] Therefore, there is an urgent need to develop a complete purification process using anion exchange high-performance liquid chromatography (HPLC) adapted to ultra-long single-stranded DNA, defining the concentration ratio of the entire mobile phase system, gradient elution parameters, temperature and elution volume range, and establishing a standardized separation scheme adapted to single-stranded DNA of different lengths. Summary of the Invention

[0009] The purpose of this invention is to provide a mobile phase system, method, and application for the separation and purification of long single-stranded DNA by anion exchange high-performance liquid chromatography, thereby solving the problems of difficulty in separating long single-stranded DNA fragments, inability to be automated and scaled up, and poor process stability in existing technologies.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] According to a first aspect of the present invention, a mobile phase system for separating and purifying long single-stranded DNA by anion exchange high-performance liquid chromatography is provided, wherein the long single-stranded DNA has a base length of 100 nt to 10 k nt; the mobile phase system comprises an equilibration buffer, a binding buffer, a washing buffer, an elution buffer, a regeneration buffer 1, and a regeneration buffer 2;

[0012] Both the equilibrium solution and the binding solution are 20 mM sodium hydroxide aqueous solution;

[0013] The washing solution is prepared by mixing 20 mM NaOH solution and 20 mM NaOH + 1.5 M NaClO4. The volume percentage of 20 mM NaOH in the washing solution is 74%~85%, and the volume percentage of 20 mM NaOH + 1.5 M NaClO4 is 15%~26%.

[0014] The eluent is a linear gradient elution system prepared by mixing 20 mM NaOH solution and 20 mM NaOH + 1.5 M NaClO4. The volume percentage of 20 mM NaOH in the eluent gradually decreases in the range of 85% to 65%, and the volume percentage of 20 mM NaOH + 1.5 M NaClO4 gradually increases in the range of 15% to 35%.

[0015] The regenerated solution 1 is a mixed solution of 20 mM NaOH and 1.5 M NaClO4;

[0016] The regenerated solution 2 is a 0.5 M sodium hydroxide aqueous solution.

[0017] According to a preferred embodiment of the present invention, the washing solution contains 76% by volume of 20 mM NaOH and 24% by volume of 20 mM NaOH + 1.5 M NaClO4.

[0018] According to a preferred embodiment of the present invention, the eluent is a linear gradient elution system with a gradient range of 76%~74% 20 mM NaOH and 24%~26% 20 mM NaOH + 1.5 M NaClO4.

[0019] According to a second aspect of the present invention, a method for separating and purifying long single-stranded DNA by anion exchange high-performance liquid chromatography is provided, using the mobile phase system described above for purification, comprising the following steps:

[0020] 1) Sample pretreatment: The crude long single-stranded DNA product was filtered through a 0.22 μm filter membrane;

[0021] 2) Column equilibration: Rinse the column with equilibration buffer for 10 column volumes;

[0022] 3) Sample loading and binding: After filtration, the sample is loaded onto the column, and then the column is washed with binding buffer for 5 column volumes;

[0023] 4) Washing: Rinse the chromatographic column with washing solution for 3 column volumes to remove short-chain nucleic acid impurities;

[0024] 5) Gradient elution: The column was washed with a linear gradient of 20 column volumes of eluent, and the eluent fractions were collected in segments. The initial volume of 20 mM NaOH accounted for 85%, and the initial volume of 20 mM NaOH + 1.5 M NaClO4 accounted for 15%. Through gradient adjustment, the final volume of 20 mM NaOH was 65%, and the final volume of 20 mM NaOH + 1.5 M NaClO4 was 35%.

[0025] 6) Column regeneration: Wash with regeneration solution 1 for 3 column volumes and regeneration solution 2 for 5 column volumes in sequence;

[0026] 7) Reequilibration: Rinse with equilibration buffer for 5 column volumes to complete a single purification cycle.

[0027] According to a preferred embodiment of the present invention, in the gradient elution step 5), the initial volume percentage of 20 mM NaOH is 76%, and the initial volume percentage of 20 mM NaOH + 1.5 M NaClO4 is 24%. Through gradient adjustment, the final volume percentage of 20 mM NaOH is 74%, and the final volume percentage of 20 mM NaOH + 1.5 M NaClO4 is 26%.

[0028] Preferably, the liquid flow rate in steps 2) to 7) is 0.5 mL / min.

[0029] Preferably, the crude long single-stranded DNA product is obtained by phage biosynthesis, and the crude product contains the target long single-stranded DNA and a longer fragment of heterochain DNA.

[0030] Preferably, the target component collected by elution is desalted and concentrated by ethanol precipitation, and the purity of the concentrated product is ≥98%.

[0031] Preferably, the column temperature of the separation column for separation and purification is set to 30℃.

[0032] According to a third aspect of the present invention, an application is provided for the anion exchange high-performance liquid chromatography method for separating and purifying long single-stranded DNA, the application including its use in the automated, large-scale preparation of long single-stranded DNA; wherein the long single-stranded DNA has a base length of 100 nt to 10 k nt.

[0033] The mobile phase system provided by the present invention comprises an equilibrium liquid, a binding liquid, a washing liquid, an eluent, a regeneration liquid 1, and a regeneration liquid 2. The function of each mobile phase is explained below:

[0034] The equilibration solution uses sodium hydroxide (NaOH) as its core component. Its function is to pre-regulate the chemical environment inside the chromatographic column, construct a high pH system, and fully protonate and positively charge the functional groups of the chromatographic stationary phase, creating conditions for the adsorption and binding of nucleic acid anions. At the same time, it can wash away residual preservation solution and impurities inside the chromatographic column.

[0035] The binding solution is also prepared solely with NaOH, ensuring that all target long single-stranded DNA and various nucleic acid impurities in the sample are stably adsorbed onto the chromatographic packing material. Neutral and positively charged non-nucleic acid impurities that are not bound to the fixation material flow directly out of the chromatographic system with the mobile phase, achieving preliminary impurity removal.

[0036] The washing solution is a mixture of NaOH and sodium perchlorate (NaClO4) to provide a low-salt environment that can selectively elute impurities with weak adsorption, including short-chain oligonucleotides, primer dimers, and small molecule salt residues, thereby effectively improving the purity of the final purified product.

[0037] The eluent is a linear gradient system composed of NaOH and NaClO4, designed to provide an environment where salt ions exhibit linear changes; during elution, the competing anion ClO4 is present. - The concentration increases gradually and linearly; the binding strength of single-stranded DNA of different lengths to the stationary phase varies, and the weaker the binding force (the shorter the fragment length), the earlier it is eluted, thus achieving the separation of ssDNA of different lengths in order of fragment length from shortest to longest.

[0038] Regeneration solution 1 is a mixture of NaOH and NaClO4, forming a high-salt, high-alkaline environment that can elute stubborn nucleic acid impurities that elute after the target long single-stranded DNA and have a strong adsorption effect, ensuring that all bound substances are removed and preparing for complete regeneration.

[0039] Regeneration solution 2 is prepared using only NaOH and is used for deep cleaning and disinfection of chromatographic packing materials, restoring the original adsorption and separation performance of the packing materials, and ensuring stable and reproducible separation and purification results for multiple batches of long single-stranded DNA.

[0040] In summary, the concentration of each component in the mobile phase system is crucial for ensuring separation efficiency; both excessively high and low concentrations will weaken the corresponding function. For example, with the washing solution: too low a NaOH concentration will result in insufficient removal of impurities; while too high a NaOH concentration will reduce the adsorption capacity of DNA to the packing material, causing the target long single-stranded DNA to be eluted prematurely, significantly reducing product recovery.

[0041] It should be understood that gradient elution step 5) includes: preparing two basic buffer solutions, solution A: 20 mM NaOH aqueous solution; solution B: 20 ​​mM NaOH + 1.5 M NaClO4 mixed solution.

[0042] The total elution volume was 20 column volumes, with a uniform linear elution rate throughout. The gradient program was set as follows:

[0043] At the start of the elution phase: the mobile phase ratio is 76% solution A + 24% solution B;

[0044] At the end of the elution phase: the mobile phase ratio smoothly and linearly changes to 74% solution A + 26% solution B;

[0045] The binary pump in liquid chromatography operates according to a linear, uniform speed program, continuously and synchronously decreasing the volume percentage of liquid A and increasing the volume percentage of liquid B within an elution time of 20 column volumes, without any step jumps or plateaus.

[0046] The chromatographic system uses a binary gradient pump to simultaneously deliver phases A and B at a constant flow rate of 0.5 mL / min. The entire elution process continuously delivers 20 column volumes of mixed mobile phase, and the eluent is collected in segments according to the chromatographic peaks to obtain single-stranded DNA components of different lengths.

[0047] As the elution process progresses, the proportion of solution B (containing a high concentration of NaClO4) slowly increases, and the competing anions ClO4 in the system... - As the concentration increases linearly, based on the difference in charge-binding force corresponding to the length of ssDNA, the short strands are eluted first, followed by the 540 nt target long single-stranded DNA, achieving separation from the 3045 nt ultra-long heterochain baseline.

[0048] Compared with the prior art, the present invention has the following significant advantages:

[0049] A dedicated mobile phase system was constructed using 20 mM NaOH and 1.5 M NaClO4 as basic reagents, and the optimal washing ratio and narrow linear elution gradient were obtained. The strong elution properties of NaClO4 were used to eliminate separation interference caused by the secondary structure and hydrophobic interaction of long single-stranded DNA. The system can stably achieve effective separation of long single-stranded DNA ranging from hundreds to thousands of nt, with a recovery yield increase of more than 15% compared with traditional NaCl-type anion exchange chromatography.

[0050] The entire liquid chromatography purification process is fully automated and highly reproducible, enabling gram-scale preparation. It completely overcomes the drawbacks of agarose / polyacrylamide gel extraction purification, such as being time-consuming and labor-intensive, having low recovery rates, being limited to microgram-scale preparation, and being prone to introducing gel and dye impurities. Magnetic bead purification also suffers from poor versatility. The purified product has a stable purity of no less than 98%, and can be directly used in gene therapy, synthetic biology, DNA information storage, and other fields without complex post-processing. The entire standardized chromatographic process is robust and controllable, significantly reducing the production cost and cycle time for industrial purification of long single-stranded DNA, and possesses extremely high commercial and industrial application value.

[0051] In summary, this invention provides a mobile phase system, purification method, and application for the separation and purification of long single-stranded DNA using anion exchange high-performance liquid chromatography (HPLC). A complete mobile phase system is constructed based on 20 mM NaOH and compounded with 1.5 M NaClO4. This mobile phase system consists of an equilibration buffer, binding buffer, washing buffer, elution buffer, regeneration buffer 1, and regeneration buffer 2. Through comparative analysis of multiple examples, the optimal washing buffer ratio of 76% 20 mM NaOH + 24% 20 mM NaOH + 1.5 M NaClO4 was determined, along with optimal washing buffer ratios of 76%~74% 20 mM NaOH and 24%~26% 20 mM NaOH + 1.5 M NaClO4. The system utilizes a narrow linear elution gradient of NaClO4 to eliminate interference from the secondary structure of nucleic acids, thanks to the strong elution effect of NaClO4. It can stably separate long single-stranded DNA ranging from hundreds to thousands of nt. Compared with the NaCl system, the liquid phase recovery rate is improved by more than 15%, and the product purity is consistently ≥98%. The entire process is highly automated and reproducible, supporting gram-scale production. It solves the technical problem that traditional purification methods are difficult to industrialize and can be widely used in the standardized preparation of long single-stranded DNA raw materials in fields such as gene therapy, synthetic biology, and DNA information storage. Attached Figure Description

[0052] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 This is a schematic diagram of monoclonal screening;

[0054] Figure 2 Schematic diagram for expanding cultivation;

[0055] Figure 3 To detect biosynthesized ssDNA using agarose gel electrophoresis;

[0056] Figure 4 Results of DNAzyme site-specific cleavage gel electrophoresis detection;

[0057] Figure 5 Electrophoresis gel image of DNA sample before gel purification;

[0058] Figure 6 Electrophoresis gel image of DNA sample before gel purification under long-wave ultraviolet light;

[0059] Figure 7 Electrophoresis gel image of DNA sample after gel purification for quality control;

[0060] Figure 8 This is the peak chromatogram of anion exchange chromatography separation and purification based on NaCl buffer in Example 3;

[0061] Figure 9 This is a 1% denaturing agarose gel electrophoresis quality control image of the recovered product based on the peak diagram in Example 3;

[0062] Figure 10 This is a quality control image of the 1% denaturing agarose gel electrophoresis of the target product after desalting and concentration in Example 3.

[0063] Figure 11 This is the peak diagram of anion exchange chromatography separation and purification based on NaClO4 buffer in Example 4;

[0064] Figure 12 This is a peak-based quality control image of the recovered product from Example 4, obtained by 15% polyacrylamide gel electrophoresis.

[0065] Figure 13 This is the peak chromatogram of anion exchange chromatography separation and purification based on NaClO4 buffer in Example 5;

[0066] Figure 14 This is a peak-based quality control image of the recovered product from Example 5, obtained by 15% polyacrylamide gel electrophoresis.

[0067] Figure 15 This is a quality control image of the 1% denaturing agarose gel electrophoresis of the target product after desalting and concentration in Example 5.

[0068] Figure 16 This is the peak chromatogram of anion exchange chromatography separation and purification based on NaClO4 buffer in Example 6;

[0069] Figure 17 This is a 1% denaturing agarose gel electrophoresis quality control image of the recovered product based on the peak diagram in Example 6;

[0070] Figure 18 This is a quality control image of the product from Example 6 after desalting and concentration using a 1% denaturing agarose gel electrophoresis.

[0071] Figure 19 This is the peak chromatogram of anion exchange chromatography separation and purification based on NaClO4 buffer in Example 7;

[0072] Figure 20 This is a 1% denaturing agarose gel electrophoresis quality control image of the recovered product based on the peak diagram in Example 7;

[0073] Figure 21 This is a quality control image of the product from Example 7 after desalting and concentration using a 1% denaturing agarose gel electrophoresis.

[0074] Figure 22This is the peak chromatogram of anion exchange chromatography separation and purification based on NaClO4 buffer in Example 8;

[0075] Figure 23 This is a 1% denaturing agarose gel electrophoresis quality control image of the recovered product based on the peak diagram in Example 8;

[0076] Figure 24 This is a quality control image of the product from Example 8 after desalting and concentration using a 1% denaturing agarose gel electrophoresis.

[0077] Figure 25 This is the peak chromatogram of anion exchange chromatography separation and purification based on NaClO4 buffer in Example 9;

[0078] Figure 26 This is a 1% denaturing agarose gel electrophoresis quality control image of the recovered product based on the peak diagram in Example 9;

[0079] Figure 27 This is a quality control image of the product from Example 9 after desalting and concentration using a 1% denaturing agarose gel electrophoresis. Detailed Implementation

[0080] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the instrument manufacturer. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0081] Example 1

[0082] In this embodiment, the method described in the reference (Jin Liu, Hongzhou Gu, Biotechnological production of ssDNA with DNA-hydrolyzing deoxyribozymes, STAR Protocols, 2021,2(2): 1000531) was used to prepare approximately 8 mg of crude long single-stranded DNA using its phage-assisted method. The fragment lengths were mainly 3045 nt and 540 nt, with the 540 nt fragment being the target band for separation and purification.

[0083] The specific steps for preparing crude long single-stranded DNA using the phage-assisted method are as follows:

[0084] Step 1: Plasmid transformation.

[0085] JM109 competent cells were thawed on ice.

[0086] Take 1 μL of plasmid and 1 μL of helper phage vcsM13, mix them thoroughly with competent cells, and incubate on ice for 30 minutes.

[0087] The sample was subjected to thermal shock conversion at 42°C for 90 seconds and immediately placed on ice for 15 minutes.

[0088] Add 500 μL of LB liquid culture medium to the sample and incubate at 37°C and 200 rpm for 1 hour in a constant temperature shaker.

[0089] Centrifuge at 5000 rpm for 2 minutes to precipitate the cells, aspirate and discard 250 μL of supernatant, and resuspend the cell pellet in the remaining culture medium.

[0090] Transformed cells were seeded at a density of 80 μL on LB agar plates containing ampicillin (100 μg / mL) and kanamycin (50 μg / mL), and incubated upside down at 37°C for 12–16 hours. Screening results are shown below. Figure 1 .

[0091] Step 2: Expand culture and extract ssDNA.

[0092] In a clean, sterilized shampoo bladder, single-clonal strains were picked from plates and inoculated into 3 L of 2×YT liquid medium. Ampicillin (100 μg / mL) and kanamycin (50 μg / mL) were added, and the medium was cultured for 12 hours at 37°C and 220 rpm in a shaker. A schematic diagram of the culture expansion is shown below. Figure 2 :

[0093] Transfer the culture to two 1.5 L centrifuge flasks, balance them, and centrifuge at 4000 g for 15 minutes at 4°C.

[0094] Collect the supernatant in a beaker, add 120 g PEG8000 and 90 g NaCl, place on a magnetic stirrer, and stir at room temperature until completely dissolved. The solution is translucent. Incubate in an ice bath for 30 minutes.

[0095] Transfer the solution to six 500 mL centrifuge bottles, balance them, and centrifuge at 16000 g for 30 minutes at 4 °C. Discard the supernatant.

[0096] Resuspend the precipitate in 30 mL TE buffer, add 4.5 μL RNase A to digest RNA, and incubate at 37°C for 10 minutes.

[0097] Centrifuge the suspension at 16,000 g for 10 minutes at 4°C to remove undissolved substances. Transfer the supernatant to a 50 mL centrifuge tube.

[0098] Add twice the volume of PPB2 solution and gently shake the mixture for 3 minutes at room temperature.

[0099] Add 1.5 times the volume of PPB3 solution, invert the container several times to mix the solution thoroughly, and then incubate in an ice bath for 10 minutes.

[0100] Centrifuge at 16000 g for 30 minutes at 4°C.

[0101] Carefully collect the supernatant into a new 50 mL centrifuge tube and discard the precipitate.

[0102] The supernatant was concentrated by ethanol precipitation to remove salt.

[0103] Dissolve the dried product in TE buffer and quantify the ssDNA concentration using Nanodrop. Verify the biosynthesized ssDNA by agarose gel electrophoresis. Quality control results are shown below. Figure 3 The quantitative results of the products are shown in Table 1.

[0104] Table 1 Nanodrop quantification of ssDNA concentration

[0105]

[0106] Step 3: DNAzyme cleavage.

[0107] Add 6000 pmol of the DNA to be cut to buffer 1 to a total volume of 40 mL.

[0108] Bath in a 90℃ water bath for 5 minutes, then cool to room temperature.

[0109] Add 80 mL of buffer 2, cut at 37°C for 16 hours, and purify and concentrate the cleavage product using ethanol precipitation.

[0110] The results of the denatured agarose gel quality inspection cutting are shown in the figure. Figure 4 .

[0111] Example 2

[0112] In this embodiment, the crude long single-stranded DNA product prepared in Example 1 was separated and purified using the traditional agarose gel purification method. Approximately 15 μg of the target fragment 540 nt long single-stranded DNA was produced, with a product purity of ≥98% and a purification recovery yield of approximately 60%.

[0113] Agarose gel purification method purification steps:

[0114] Preparation of 1% denaturing agarose gel: Weigh 1 g of agarose powder, add 100 mL of 1x TAE buffer, heat in a microwave oven, boil repeatedly 5 times until the solution is clear and transparent with no visible particles, add 12 g of urea until dissolved, place the conical flask to cool to room temperature (about 50~60℃) and add 5 μL of nucleic acid dye, pour into the gel casting tank, insert the comb, and let stand at room temperature for 30~60 minutes until the gel is completely solidified.

[0115] Preparation of long single-stranded DNA samples: Add an equal volume of DNA sample to 2x denaturing loading buffer and mix. Denature at 95°C for 2 minutes and immediately place on ice to cool.

[0116] Sample loading and electrophoresis: Place the gel in the electrophoresis tank, add 1x TAE as electrophoresis buffer, add approximately 150 μg of DNA sample, and electrophoresis at 80 V for 330 minutes. See the electrophoresis gel image below. Figure 5 .

[0117] Gel cutting: Under long-wave ultraviolet light, use a clean blade to cut the target band and place it into a pre-weighed 15 mL centrifuge tube. The electrophoresis gel image under long-wave ultraviolet light is shown below. Figure 6 .

[0118] Reagent Recovery: Weigh the gel product. Add membrane binding buffer at a ratio of 2 mL per 1 g of gel. Incubate in a 50°C water bath for 30–50 minutes until completely dissolved. Remove the sample and cool to room temperature. Add the gel solution to a centrifuge adsorption column (place the adsorption column in a collection tube). Incubate at room temperature for 2 minutes to allow DNA to fully bind to the silica membrane. Centrifuge at 11,000 rpm for 1 minute, discard the waste liquid in the collection tube, and repeat the process until all the gel solution is added to the same adsorption column. Add membrane washing buffer to the adsorption column. Centrifuge at 11,000 rpm for 1 minute, discard the waste liquid, and repeat the washing process once, followed by centrifugation once with an empty column. Open the cap and allow to dry at room temperature for 2–3 minutes. Transfer the adsorption column to a new 1.5 mL centrifuge tube. Add 50 μL of sterile water dropwise to the center of the adsorption membrane. Incubate at room temperature for 2 minutes, then centrifuge at 11,000 rpm for 1 minute to obtain approximately 15 μg of purified long single-stranded DNA, with a purification recovery yield of approximately 60%.

[0119] Quality control: The purified product, consisting of 540 nt long single-stranded DNA, was subjected to denaturing agarose gel electrophoresis for quality control. The results are shown in the table below. Figure 7 Purity analysis was performed using ImageJ software, and the purity was ≥98%.

[0120] Example 3

[0121] This embodiment selects the NaCl-eluting-based anion exchange chromatography method from existing technologies (see patents CN117143165B and CN121591819A) as a comparative example of the NaClO4 elution system of this invention. This embodiment separates and purifies the crude long single-stranded DNA product prepared in Example 1. In this embodiment, approximately 9.4 μg of the target fragment (540 nt long single-stranded DNA) was produced, with a product purity ≥98% and a target fragment yield of approximately 25.8%.

[0122] The purification steps of anion exchange chromatography are as follows:

[0123] Sample preparation: DNA samples were filtered using a 0.22 μm needle filter.

[0124] Column equilibration: Rinse the column with 10 column volumes of 10 mM NaOH equilibration solution.

[0125] Sample loading: Approximately 240 μg of pretreated DNA sample is slowly and evenly loaded into the chromatographic column through the injection tubing.

[0126] Binding: Rinse the column with 5 column volumes of 10 mM NaOH binding solution.

[0127] Washing: Use a washing solution made by mixing 10 mM NaOH equilibration solution and 10 mM NaOH + 2 M NaCl regeneration solution in a certain ratio to wash the chromatographic column for 3 column volumes.

[0128] Elution: The column was rinsed with 20 column volumes of elution buffer by mixing 10 mM NaOH equilibration solution and 10 mM NaOH + 2 M NaCl regeneration solution in a certain linear gradient ratio.

[0129] Regeneration 1: Rinse the column with 10 mM NaOH + 2 M NaCl regeneration solution 1 for 3 column volumes.

[0130] Regeneration 2: Rinse the column with 0.5 M NaOH regeneration solution 2 for 5 column volumes.

[0131] Equilibration: Rinse the column with 5 column volumes of 10 mM NaOH equilibration buffer to prepare for the next round of sample purification.

[0132] The specific purification procedure is shown in the table below:

[0133] Table 2 Anion exchange chromatography separation and purification protocol for 540 nt DNA samples

[0134]

[0135] The purification peak chromatogram results are shown below. Figure 8 .

[0136] Quality control of recovered products: The recovered products were subjected to 1% denaturing agarose gel electrophoresis for quality control. The results are shown in the table below. Figure 9 :

[0137] The gel imaging results showed that the target fragment, a 540 nt long single-stranded DNA, was isolated from the recovered product of peak F1.

[0138] Product concentration and quality control: The purified products ①, ②, and ③ containing the target band were selected for ethanol precipitation, desalting, and concentration. The purified products were then subjected to 1% denaturing agarose gel electrophoresis for quality control. The results are shown below. Figure 10 Approximately 9.4 μg of the target long single-stranded DNA was obtained, with an average product purity of ≥98% and a yield of approximately 25.8%.

[0139] Example 4

[0140] This embodiment uses anion exchange chromatography based on a NaClO4 elution system to separate and purify the crude long single-stranded DNA product prepared in Example 1. The input amount of crude product is approximately 240.0 μg, of which approximately 36.4 μg is the target fragment of 540 nt long single-stranded DNA. The final yield of the target fragment of 540 nt long single-stranded DNA is 0 μg.

[0141] The purification steps of anion exchange chromatography are as follows:

[0142] Sample preparation: DNA samples were filtered using a 0.22 μm needle filter.

[0143] Column equilibration: Rinse the column with 10 column volumes of 20 mM NaOH equilibration solution.

[0144] Sample loading: Approximately 240 μg of pretreated DNA sample is slowly and evenly loaded into the chromatographic column through the injection tubing.

[0145] Binding: Rinse the column with 5 column volumes of 20 mM NaOH binding solution.

[0146] Washing: Rinse the column for 3 column volumes using a washing solution made of 85% 20 mM NaOH equilibration solution and 15% 20 mM NaOH + 1.5 M NaClO4 regeneration solution.

[0147] Elution: The column was rinsed with an eluent consisting of 85%~65% 20 mM NaOH equilibration buffer and 15%~35% 20 mM NaOH + 1.5 M NaClO4 regeneration buffer in a linear gradient ratio for 20 column volumes.

[0148] Regeneration 1: Rinse the column with 20 mM NaOH + 1.5 M NaClO4 regeneration solution 1 for 3 column volumes.

[0149] Regeneration 2: Rinse the column with 0.5 M NaOH regeneration solution 2 for 5 column volumes.

[0150] Equilibration: Rinse the column with 5 column volumes of 20 mM NaOH equilibration buffer to prepare for the next round of sample purification.

[0151] The specific purification procedure is shown in the table below:

[0152] Table 3. DNA Sample Anion Exchange Chromatography Separation and Purification Scheme

[0153]

[0154] The purification peak chromatogram results are shown below. Figure 11 .

[0155] Quality control of recovered products: The recovered products were subjected to 15% denaturing polyacrylamide gel electrophoresis for quality control. The results are shown in the table below. Figure 12 .

[0156] The gel electrophoresis results showed that the target fragment was not separated in the recovered products of peaks F1 and F2, so no further desalting and concentration were performed.

[0157] Example 5

[0158] This embodiment uses anion exchange chromatography based on a NaClO4 elution system to separate and purify the crude long single-stranded DNA product prepared in Example 1. The crude product input is approximately 240.0 μg, of which approximately 36.4 μg is the target fragment of 540 nt long single-stranded DNA. The specific purification process in this embodiment is an optimization of the purification process in Example 4. The optimization result is that approximately 6.0 μg of the target fragment of 540 nt long single-stranded DNA is produced, the product purity is ≥98%, and the yield is approximately 16.5%.

[0159] The purification steps of anion exchange chromatography are as follows:

[0160] Sample preparation: DNA samples were filtered using a 0.22 μm needle filter.

[0161] Column equilibration: Rinse the column with 10 column volumes of 20 mM NaOH equilibration solution.

[0162] Sample loading: Approximately 240 μg of pretreated DNA sample is slowly and evenly loaded into the chromatographic column through the injection tubing.

[0163] Binding: Rinse the column with 5 column volumes of 20 mM NaOH binding solution.

[0164] Washing: Rinse the column for 3 column volumes using a washing solution made of 80% 20 mM NaOH equilibration solution and 20% 20 mM NaOH + 1.5 M NaClO4 regeneration solution.

[0165] Elution: The column was rinsed with an eluent consisting of 80%~70% 20 mM NaOH equilibration buffer and 20%~30% 20 mM NaOH + 1.5 M NaClO4 regeneration buffer in a linear gradient ratio for 20 column volumes.

[0166] Regeneration 1: Rinse the column with 20 mM NaOH + 1.5 M NaClO4 regeneration solution 1 for 3 column volumes.

[0167] Regeneration 2: Rinse the column with 0.5 M NaOH regeneration solution 2 for 5 column volumes.

[0168] Equilibration: Rinse the column with 5 column volumes of 20 mM NaOH equilibration buffer to prepare for the next round of sample purification.

[0169] The specific purification procedure is shown in the table below:

[0170] Table 4. Anion exchange chromatography separation and purification scheme for DNA samples

[0171]

[0172] The purification peak chromatogram results are shown below. Figure 13 .

[0173] Quality control of recovered products: The recovered products were subjected to 15% denaturing polyacrylamide gel electrophoresis for quality control. The results are shown in the table below. Figure 14 .

[0174] The gel imaging results showed that among the recovered products of peaks F1, F2 and F3, the target fragment of 540 nt long single-stranded DNA was separated in the recovered products of F2 and F3.

[0175] Product concentration and quality control: Purified products ④, ⑤, and ⑥ containing the target band were selected for ethanol precipitation, desalting, and concentration. The purified products were then subjected to 1% denaturing agarose gel electrophoresis for quality control. Results are shown below. Figure 15 Approximately 6.0 μg of the target long single-stranded DNA was obtained, with an average product purity of ≥98% and a yield of approximately 16.5%.

[0176] Example 6

[0177] This embodiment uses anion exchange chromatography based on a NaClO4 elution system to separate and purify the crude long single-stranded DNA product prepared in Example 1. The crude product input is approximately 240.0 μg, of which approximately 36.4 μg is the target fragment of 540 nt long single-stranded DNA. The specific purification process in this embodiment is an optimization of the purification process in Example 5. The optimization result is that approximately 9.3 μg of the target fragment of 540 nt long single-stranded DNA is produced, the product purity is ≥98%, and the yield is approximately 25.5%.

[0178] The purification steps of anion exchange chromatography are as follows:

[0179] Sample preparation: DNA samples were filtered using a 0.22 μm needle filter.

[0180] Column equilibration: Rinse the column with 10 column volumes of 20 mM NaOH equilibration solution.

[0181] Sample loading: Approximately 240 μg of pretreated DNA sample is slowly and evenly loaded into the chromatographic column through the injection tubing.

[0182] Binding: Rinse the column with 5 column volumes of 20 mM NaOH binding solution.

[0183] Washing: Rinse the column for 3 column volumes using a washing solution made of 80% 20 mM NaOH equilibration solution and 20% 20 mM NaOH + 1.5 M NaClO4 regeneration solution.

[0184] Elution: The column was rinsed with an eluent consisting of 80%~74% 20 mM NaOH equilibration buffer and 20%~26% 20 mM NaOH + 1.5 M NaClO4 regeneration buffer in a linear gradient ratio for 20 column volumes.

[0185] Regeneration 1: Rinse the column with 20 mM NaOH + 1.5 M NaClO4 regeneration solution 1 for 3 column volumes.

[0186] Regeneration 2: Rinse the column with 0.5 M NaOH regeneration solution 2 for 5 column volumes.

[0187] Equilibration: Rinse the column with 5 column volumes of 20 mM NaOH equilibration buffer to prepare for the next round of sample purification.

[0188] The specific purification procedure is shown in the table below:

[0189] Table 5. DNA Sample Anion Exchange Chromatography Separation and Purification Scheme

[0190]

[0191] The purification peak chromatogram results are shown below. Figure 16 .

[0192] Quality control of recovered products: The recovered products were subjected to 1% denaturing agarose gel electrophoresis for quality control. The results are shown in the table below. Figure 17 .

[0193] The gel imaging results showed that the target fragment, a 540 nt long single-stranded DNA, was isolated from the recovered products of peaks F1 and F2.

[0194] Product concentration and quality control: Purified products ②, ③, ④, ⑤, ⑥, and ⑦ containing the target band were selected for ethanol precipitation, desalting, and concentration. The purified products were then subjected to 1% denaturing agarose gel electrophoresis for quality control. Results are shown below. Figure 18 Given that product number 7 contained a small amount of non-target bands, after removing product number 7, approximately 9.3 μg of the target long single-stranded DNA was obtained, with an average product purity of ≥98% and a yield of approximately 25.5%.

[0195] Example 7

[0196] This embodiment uses anion exchange chromatography based on a NaClO4 elution system to separate and purify the crude long single-stranded DNA product prepared in Example 1. The crude product input is approximately 240.0 μg, of which approximately 36.4 μg is the target fragment of 540 nt long single-stranded DNA. The specific purification process in this embodiment is an optimization of the purification process in Example 5. The optimization result is that approximately 14.0 μg of the target fragment of 540 nt long single-stranded DNA is produced, the product purity is ≥98%, and the yield is approximately 38.6%.

[0197] The purification steps for anion exchange chromatography are as follows:

[0198] Sample preparation: DNA samples were filtered using a 0.22 μm needle filter.

[0199] Column equilibration: Rinse the column with 10 column volumes of 20 mM NaOH equilibration solution.

[0200] Sample loading: Approximately 240 μg of pretreated DNA sample is slowly and evenly loaded into the chromatographic column through the injection tubing.

[0201] Binding: Rinse the column with 5 column volumes of 20 mM NaOH binding solution.

[0202] Washing: The column was washed with a mixture of 76% 20 mM NaOH equilibration solution and 24% 20 mM NaOH + 1.5 M NaClO4 regeneration solution for 3 column volumes.

[0203] Elution: The column was rinsed with an eluent consisting of 76%–70% 20 mM NaOH equilibration buffer and a linear gradient of 24%–30% 20 mM NaOH + 1.5 M NaClO4 for 20 column volumes.

[0204] Regeneration 1: Rinse the column with 20 mM NaOH + 1.5 M NaClO4 regeneration solution 1 for 3 column volumes.

[0205] Regeneration 2: Rinse the column with 0.5 M NaOH regeneration solution 2 for 5 column volumes.

[0206] Equilibration: Rinse the column with 5 column volumes of 20 mM NaOH equilibration buffer to prepare for the next round of sample purification.

[0207] The specific purification procedure is shown in the table below:

[0208] Table 6. DNA Sample Anion Exchange Chromatography Separation and Purification Scheme

[0209]

[0210] The purification peak chromatogram results are shown below. Figure 19 :

[0211] Quality control of recovered products: The recovered products were subjected to 1% denaturing agarose gel electrophoresis for quality control. The results are shown in the table below. Figure 20 :

[0212] The gel electrophoresis results showed that the target fragment of 540 nt long single-stranded DNA was isolated from the recovered products of peaks F2 and F3.

[0213] Product Concentration and Quality Inspection: The purified products containing the target band (⑤, ⑥, ⑦, ⑧, and ⑨) were selected for ethanol precipitation, desalting, and concentration. The purified products were then subjected to 1% denaturing agarose gel electrophoresis for quality inspection. The results are shown below. Figure 21 Approximately 14.0 μg of the target long single-stranded DNA was obtained, with an average product purity of ≥98% and a yield of approximately 38.6%.

[0214] Example 8

[0215] This embodiment uses anion exchange chromatography based on a NaClO4 elution system to separate and purify the crude long single-stranded DNA product prepared in Example 1. The crude product input is approximately 240.0 μg, of which approximately 36.4 μg is the target fragment of 540 nt long single-stranded DNA. The specific purification process in this embodiment is an optimization of the purification processes in Examples 5, 6, and 7. The optimization result is that approximately 15.1 μg of the target fragment of 540 nt long single-stranded DNA is produced, the product purity is ≥98%, and the yield is approximately 41.6%.

[0216] The purification steps of anion exchange chromatography are as follows:

[0217] Sample preparation: DNA samples were filtered using a 0.22 μm needle filter.

[0218] Column equilibration: Rinse the column with 10 column volumes of 20 mM NaOH equilibration solution.

[0219] Sample loading: Approximately 240 μg of pretreated DNA sample is slowly and evenly loaded into the chromatographic column through the injection tubing.

[0220] Binding: Rinse the column with 5 column volumes of 20 mM NaOH binding solution.

[0221] Washing: The column was washed with a mixture of 76% 20 mM NaOH equilibration solution and 24% 20 mM NaOH + 1.5 M NaClO4 regeneration solution for 3 column volumes.

[0222] Elution: The column was rinsed with an eluent consisting of a linear gradient of 76%–74% 20 mM NaOH equilibration buffer and 24%–26% 20 mM NaOH + 1.5 M NaClO4 for 20 column volumes.

[0223] Regeneration 1: Rinse the column with 20 mM NaOH + 1.5 M NaClO4 regeneration solution 1 for 3 column volumes.

[0224] Regeneration 2: Rinse the column with 0.5 M NaOH regeneration solution 2 for 5 column volumes.

[0225] Equilibration: Rinse the column with 5 column volumes of 20 mM NaOH equilibration buffer to prepare for the next round of sample purification.

[0226] The specific purification procedure is shown in the table below:

[0227] Table 7. DNA Sample Anion Exchange Chromatography Separation and Purification Scheme

[0228]

[0229] The purification peak chromatogram results are shown below. Figure 22 .

[0230] Quality control of recovered products: The recovered products were subjected to 1% denaturing agarose gel electrophoresis for quality control. The results are shown in the table below. Figure 23 .

[0231] The gel imaging results showed that the target fragment of 540 nt long single-stranded DNA was isolated from the recovered products of peaks F1, F2, and F3.

[0232] Product concentration and quality control: The purified product containing the target band was selected for ethanol precipitation, desalting and concentration, and then subjected to 1% denaturing agarose gel electrophoresis for quality control again. The results are shown in the figure. Figure 24 Approximately 15.1 μg of the target long single-stranded DNA was obtained, with an average product purity of ≥98% and a yield of approximately 41.6%.

[0233] To construct a standardized purification process for long single-stranded DNA with stable separation effect and good reproducibility, this invention verified existing separation and purification methods in Examples 2-3, adopted a NaClO4-based mobile phase system in Example 4, and further conducted multiple sets of comparative optimizations on key process parameters such as washing solution ratio and linear elution gradient in Examples 5-8.

[0234] As the optimal solution, the washing solution is prepared by mixing 76% by volume of 20 mM NaOH solution and 24% by volume of 20 mM NaOH + 1.5 M NaClO4 solution. As in Examples 7 and 8, this washing solution ratio is better for eluting and removing short-chain impurities than Example 4 (washing solution 85% 20 mM NaOH + 15% high-salt buffer) and Example 5 (washing solution 80% 20 mM NaOH + 20% high-salt buffer), which can reduce the co-elution of impurities and target fragments and significantly improve the product recovery rate.

[0235] As the optimal solution, the eluent adopts a linear gradient elution mode: in the initial stage of elution, the mobile phase consists of 76% (v / v) 20 mM NaOH solution and 24% (v / v) 20 mM NaOH + 1.5 M NaClO4 solution, and at the end of elution, it linearly transitions to 74% (v / v) 20 mM NaOH solution and 26% (v / v) 20 mM NaOH + 1.5 M NaClO4 solution. Example 8 uses this narrow-range linear gradient, which, compared with the wide gradient in Example 5 (80%~70% 20 mM NaOH, 20%~30% high-salt buffer) and the gradient in Example 6 (80%~74% 20 mM NaOH, 20%~26% high-salt buffer), can achieve more complete baseline separation between the 540 nt target fragment and the 3045 nt heterochain, and the target product recovery yield is increased to 41.6%, with the best purification effect.

[0236] Example 9

[0237] This embodiment uses a mixture of 1300 nt, 3000 nt, and 4300 nt long single-stranded DNA as the purification sample to verify the optimal solution of Example 8. The crude product input of the long single-stranded DNA mixture is approximately 220.0 μg, of which approximately 50.0 μg is the target fragment 1300 nt long single-stranded DNA. The specific purification process in this embodiment is the purification process in Example 8. The purification result is approximately 21.0 μg of the target fragment 1300 nt long single-stranded DNA, with a product purity ≥98% and a yield of approximately 42.0%.

[0238] The purification steps of anion exchange chromatography are as follows:

[0239] Sample preparation: DNA samples were filtered using a 0.22 μm needle filter.

[0240] Column equilibration: Rinse the column with 10 column volumes of 20 mM NaOH equilibration solution.

[0241] Sample loading: Approximately 220 μg of pretreated DNA sample is slowly and evenly loaded into the chromatographic column through the injection tubing.

[0242] Binding: Rinse the column with 5 column volumes of 20 mM NaOH binding solution.

[0243] Washing: The column was washed with a mixture of 76% 20 mM NaOH equilibration solution and 24% 20 mM NaOH + 1.5 M NaClO4 regeneration solution for 3 column volumes.

[0244] Elution: The column was rinsed with an eluent consisting of a linear gradient of 76%–74% 20 mM NaOH equilibration buffer and 24%–26% 20 mM NaOH + 1.5 M NaClO4 for 20 column volumes.

[0245] Regeneration 1: Rinse the column with 20 mM NaOH + 1.5 M NaClO4 regeneration solution 1 for 3 column volumes.

[0246] Regeneration 2: Rinse the column with 0.5 M NaOH regeneration solution 2 for 5 column volumes.

[0247] Equilibration: Rinse the column with 5 column volumes of 20 mM NaOH equilibration buffer to prepare for the next round of sample purification.

[0248] The specific purification procedure is shown in the table below:

[0249] Table 8. DNA Sample Anion Exchange Chromatography Separation and Purification Scheme

[0250]

[0251] The purification peak chromatogram results are shown below. Figure 25 .

[0252] Quality control of recovered products: The recovered products were subjected to 1% denaturing agarose gel electrophoresis for quality control. The results are shown in the table below. Figure 26 .

[0253] The gel imaging results showed that the recovered product of peak F1 was the target fragment, a 1300 nt long single-stranded DNA.

[0254] Product concentration and quality control: The purified product containing the target band was selected for ethanol precipitation, desalting and concentration, and then subjected to 1% denaturing agarose gel electrophoresis for quality control again. The results are shown in the figure. Figure 27 The target fragment, a 1300 nt long single-stranded DNA, was produced in a yield of approximately 21.0 μg, with a purity of ≥98% and a yield of approximately 42.0%.

[0255] This embodiment, based on Example 8, uses mixtures of 1300 nt, 3000 nt, and 4300 nt long single-stranded DNA to validate the optimal purification scheme of Example 8. The results of producing the target fragment of 1300 nt long single-stranded DNA indicate that Example 8, as the optimal scheme, has the characteristics of stable separation effect and good reproducibility.

[0256] In summary, while the above embodiments show that, at the same product purity, this method only achieves a 41% purification recovery rate compared to the 60% recovery rate of the traditional denaturing agarose gel purification method, which is currently relatively low, its advantages lie in its high adaptability, high automation, high reproducibility, ease of scaling up production, and ability to achieve gram-scale production. This is a significant advantage over the microgram-scale production of gel excision purification, and the recovery rate can be further improved through process optimization. Furthermore, at the same product purity, this method improves the purification recovery rate by at least 15% compared to anion exchange high-performance liquid chromatography based on a NaCl buffer system, effectively reducing the loss of purification raw materials. This method is also scalable; in addition to purifying mixtures of 540 nt and 3000 nt long single-stranded DNA, it can also separate the 1300 nt target fragment from mixtures of long single-stranded DNA, including but not limited to 1300 nt, 3000 nt, and 4300 nt. In conclusion, the mobile phase system of this invention can stably separate hundreds to thousands of nt long single-stranded DNA.

[0257] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A mobile phase system for the separation and purification of long single-stranded DNA by anion exchange high-performance liquid chromatography, characterized in that, The long single-stranded DNA has a base length of 100 nt to 10 k nt; the mobile phase system consists of equilibration buffer, binding buffer, washing buffer, elution buffer, regeneration buffer 1, and regeneration buffer 2; Both the equilibrium solution and the binding solution are 20 mM sodium hydroxide aqueous solution; The washing solution is prepared by mixing 20 mM NaOH solution and 20 mM NaOH + 1.5 M NaClO4. The volume percentage of 20 mM NaOH in the washing solution is 74%~85%, and the volume percentage of 20 mM NaOH + 1.5 M NaClO4 is 15%~26%. The eluent is a linear gradient elution system prepared by mixing 20 mM NaOH solution and 20 mM NaOH + 1.5 M NaClO4. The volume percentage of 20 mM NaOH in the eluent gradually decreases in the range of 85% to 65%, and the volume percentage of 20 mM NaOH + 1.5 M NaClO4 gradually increases in the range of 15% to 35%. The regenerated solution 1 is a mixed solution of 20 mM NaOH and 1.5 M NaClO4; The regenerated solution 2 is a 0.5 M sodium hydroxide aqueous solution.

2. The mobile phase system according to claim 1, characterized in that, The volume percentage of 20 mM NaOH in the washing solution was 76%, and the volume percentage of 20 mM NaOH + 1.5 M NaClO4 was 24%.

3. The mobile phase system according to claim 1, characterized in that, The eluent is a linear gradient elution system with a gradient range of 76%~74% 20 mM NaOH and 24%~26% 20 mM NaOH + 1.5 M NaClO4.

4. A method for separating and purifying long single-stranded DNA by anion exchange high-performance liquid chromatography, characterized in that, Purification using the mobile phase system described in any one of claims 1 to 3 includes the following steps: 1) Sample pretreatment: The crude long single-stranded DNA product was filtered through a 0.22 μm filter membrane; 2) Column equilibration: Rinse the column with equilibration buffer for 10 column volumes; 3) Sample loading and binding: After filtration, the sample is loaded onto the column, and then the column is washed with binding buffer for 5 column volumes; 4) Washing: Rinse the chromatographic column with washing solution for 3 column volumes to remove short-chain nucleic acid impurities; 5) Gradient elution: The column was washed with a linear gradient of 20 column volumes of eluent, and the eluent fractions were collected in segments. The initial volume of 20 mM NaOH accounted for 85%, and the initial volume of 20 mM NaOH + 1.5 M NaClO4 accounted for 15%. Through gradient adjustment, the final volume of 20 mM NaOH was 65%, and the final volume of 20 mM NaOH + 1.5 M NaClO4 was 35%. 6) Column regeneration: Wash with regeneration solution 1 for 3 column volumes and regeneration solution 2 for 5 column volumes in sequence; 7) Reequilibration: Rinse with equilibration buffer for 5 column volumes to complete a single purification cycle.

5. The method for separating and purifying long single-stranded DNA according to claim 4, characterized in that, In gradient elution step 5), the initial volume percentage of 20 mM NaOH is 76%, and the initial volume percentage of 20 mM NaOH + 1.5 M NaClO4 is 24%. Through gradient adjustment, the final volume percentage of 20 mM NaOH is 74%, and the final volume percentage of 20 mM NaOH + 1.5 M NaClO4 is 26%.

6. The method for separating and purifying long single-stranded DNA according to claim 4, characterized in that, Steps 2) through 7) have a liquid flow rate of 0.5 mL / min throughout.

7. The method for separating and purifying long single-stranded DNA according to claim 4, characterized in that, The crude long single-stranded DNA product is obtained by phage biosynthesis and contains the target long single-stranded DNA and a longer fragment of heterochain DNA.

8. The method for separating and purifying long single-stranded DNA according to claim 4, characterized in that, The column temperature for separation and purification was set to 30℃.

9. An application of anion exchange high-performance liquid chromatography for the separation and purification of long single-stranded DNA according to any one of claims 4 to 8, characterized in that, The applications include the automated, large-scale preparation of long single-stranded DNA.

10. The application according to claim 9, characterized in that, The long single-stranded DNA has a base length of 100 nt to 10 knt.

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