Device and method for eluting, purifying and recovering oversized DNA based on pulsed field gel electrophoresis

Abstract

The invention relates to a device and a method for eluting, purifying and recovering oversized DNA (deoxyribonucleic acid) based on pulsed field gel electrophoresis, which are used for efficiently recovering complete DNA molecules from hundred kb level to Mb level. The device comprises an electrophoresis protection chamber with a polygonal container structure and a sample recovery chamber, the side edge of the protection chamber is provided with a semi-permeable membrane for current to pass through, and the recovery chamber is detachably placed in the center and comprises the semi-permeable membrane; the detachable and replaceable magnesium sheet is arranged in the protection chamber and is used for ionizing magnesium ions to promote DNA (Deoxyribose Nucleic Acid) compression; the method comprises the following steps: preparing a biological sample into an agarose gel block, cracking and cleaning the agarose gel block, placing the agarose gel block in a device, and adding a recovery protection solution containing PEG; under the condition of pulsed field gel electrophoresis, DNA migrates out of a gel block and is combined with magnesium ions to be compressed into spheres, and the spheres are collected in a recovery chamber after charges are neutralized. Compared with the prior art, the method provided by the invention overcomes the defects of low product purity and tedious steps of a traditional recovery method, realizes high-purity and high-efficiency integrated recovery, and is suitable for genomics research and biotechnology application.

Description

Technical Field

[0001] This invention relates to nucleic acid separation and purification technology in the field of biotechnology, and in particular to an apparatus and method for eluting and purifying ultra-large DNA based on pulsed field gel electrophoresis. Background Technology

[0002] With the rapid development of genome sequencing technology and synthetic genomics, the in vitro recovery and purification of ultra-large DNA ranging from hundreds of kb to Mb has become a crucial link connecting the "blueprint" and "function" of the genome, enabling researchers to directly manipulate genetic material on a larger scale. Currently, the mainstream method relies on pulsed-field gel electrophoresis (PFGE) technology, such as separating DNA by PFGE followed by digestion with agarose enzyme (as described in the two-dimensional electrophoresis method in CN102586227A), or using an electroelution device for field-reversed gel electrophoresis recovery. However, these methods have revealed significant limitations in practical applications: although CN102586227A optimizes the separation effect through two-dimensional PFGE (FIGE and CHEF modes), the recovery step requires digestion of low-melting-point agarose gel with agarose enzyme, resulting in products containing high concentrations of salt ions, polysaccharides, and enzyme residues, leading to low purity and a lengthy process (approximately 3 days per recovery), which is not conducive to subsequent operations.

[0003] Further analysis of existing technologies reveals that CN1479790A discloses a rapid microbial typing method that uses a PFGE microdevice to shorten electrophoresis time to 2.5-7 hours and emphasizes enzyme-free DNA sample preparation to reduce costs. However, this method focuses on microbial typing and does not solve the purification problem in the recovery of large DNA samples; after electrophoretic separation, it still relies on traditional recovery methods, which cannot avoid product contamination. Meanwhile, while field reverse gel electrophoresis (as mentioned in CN102586227 A) attempts to improve recovery efficiency, DNA is easily blocked in the gel, resulting in low recovery volume and detection only through Southern hybridization, making it impractical (Viovy et al., Electrophoresis, 1992). The aforementioned reference is: Viovy, JL, et al., Irreversible Trapping of DNA during Crossed-Field Gel-Electrophoresis. Electrophoresis, 1992. 13(1-2): p. 1-6.

[0004] In summary, the core problem with existing technologies lies in the fact that pulsed-field gel electrophoresis combined with enzymatic digestion or electroelution suffers from low recovery efficiency, insufficient product purity, and complex processes when recovering ultra-large DNA. This directly restricts the application of ultra-large DNA in functional genomics, necessitating a highly efficient, pure, and simplified recovery and purification technology. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an apparatus and method for purifying and recovering ultra-large DNA based on pulsed field gel electrophoresis. The in vitro purification and recovery of ultra-large DNA is simple to operate, the entire recovery process is short, the total amount of ultra-large DNA fragments obtained in one recovery is large, the integrity is high, and it can be stably stored for a long time.

[0006] The objective of this invention can be achieved through the following technical solutions: The first aspect of this invention provides an apparatus for eluting, purifying, and recovering ultra-large DNA based on pulsed-field gel electrophoresis, comprising an electrophoresis protection chamber, a sample recovery chamber, and a removable and replaceable magnesium plate. The electrophoresis protection chamber is a polygonal container structure with a semi-permeable membrane on its side. The electrophoresis protection chamber is used to contain the electrophoresis protection solution and allow the current to pass through the semi-permeable membrane during pulsed field gel electrophoresis. The sample recovery chamber is a polygonal container structure with a semi-permeable membrane in the middle of its side. The sample recovery chamber is detachably placed in the center of the electrophoresis protection chamber, and its side is parallel to the corresponding side of the electrophoresis protection chamber, so that the current passes through evenly. The sample recovery chamber is used to place the ultra-large DNA sample gel block to be recovered and add electrophoresis protection solution. A removable and replaceable magnesium sheet is provided in the electrophoresis protection chamber. The removable and replaceable magnesium sheet is parallel to the side of the sample recovery chamber and maintains a predetermined distance. It is used to ionize magnesium ions during electrophoresis to promote the compression and protection of ultra-large DNA.

[0007] Furthermore, the semi-permeable membranes of the electrophoresis protection chamber and the sample recovery chamber are made of materials that allow ions and small molecules to pass through but block the permeation of ultra-large DNA, thereby allowing ultra-large DNA to be retained in the sample recovery chamber during the electroelution process.

[0008] Furthermore, the electrophoresis protection chamber is a container with a hexagonal bottom, and the sample recovery chamber is a container with a trapezoidal bottom.

[0009] Furthermore, the electrophoresis protection chamber, sample recovery chamber, and detachable and replaceable magnesium sheet are all independent components, which are assembled before use to recover ultra-large DNA samples; The electrophoresis protection chamber and sample recovery chamber are immersed in ultrapure water or other solutions for long-term storage after use, and can be reused multiple times.

[0010] A second aspect of this invention provides a method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis, comprising the following steps: S1. After culturing and concentrating biological samples containing ultra-large DNA fragments, pulsed field gel electrophoresis blocks were prepared using low-melting-point agarose. The gel blocks were then subjected to lysis and washing to expose the ultra-large DNA. S2. Assemble the above-mentioned device for elution, purification and recovery of ultra-large DNA based on pulsed field gel electrophoresis. Place the processed gel block in the sample recovery chamber and place it close to the side semi-permeable membrane. Add recovery protection solution containing PEG polymer to the sample recovery chamber and the electrophoresis protection chamber so that the liquid surface submerges the pulsed field gel electrophoresis gel block. Then place the entire device in the pulsed field gel electrophoresis tank and add electrophoresis buffer. S3. Electrophoresis is performed under pulsed field gel electrophoresis conditions, which allows the super-large DNA to migrate out of the gel block and bind with magnesium ions ionized by the removable and replaceable magnesium sheet. Under the action of the recovery protective solution, the DNA is compressed into a spherical shape, thereby neutralizing the charge and stopping the migration, and is collected in the sample recovery chamber. S4. Draw the solution containing compressed, oversized DNA from the sample recovery chamber.

[0011] Further, in S1, the lysis treatment involves digesting the gel block at 50°C for 12 to 48 hours using a lysis buffer containing proteinase K, wherein the lysis buffer contains 500 mM EDTA at pH 8.0, 1 wt% sodium lauroyl sarcosinate and 200 μg / mL proteinase K. The washing process involves washing the digested gel block multiple times at room temperature using a pH 8.0 TE buffer for 30 to 60 minutes each time to ensure the integrity and purity of the ultra-large DNA and to prevent degradation.

[0012] Furthermore, in S3, the pulsed field gel electrophoresis conditions specifically include: depending on the size of the recovered DNA sample, the electrophoresis temperature is controlled at 7-14℃, the electric field strength is set to 2-6 V / cm, the pulsed electric field conversion time is 30 seconds to 500 seconds, the pulsed electric field conversion angle is 106°-120°, and the total electrophoresis time is adjusted between 1 hour and 12 hours according to the size of the target DNA fragment, so that the ultra-large DNA fragment can migrate out of the gel block and undergo strand-globulin conversion.

[0013] Furthermore, in S3, the pulsed electric field switching time is specifically adjusted according to the size of the ultra-large DNA fragment to be recovered, specifically as follows: When the recovered DNA fragments are in the hundreds of kb range, the pulse turn-off time is set to 30-90 seconds; When the recovered DNA fragments are in the Mb range, the pulse turn-off time is set to 90-500 seconds to accommodate the migration characteristics of DNA fragments of different sizes in the pulse field, thereby optimizing separation efficiency and recovery yield.

[0014] Furthermore, in S2, the recovered protective liquid contains 15 wt% PEG polymer; the degree of polymerization of PEG ranges from 3000 to 20000.

[0015] The PEG polymer is dissolved in 0.5×TBE buffer. The PEG polymer is used to promote the chain-to-sphere transformation of super-large DNA after binding with magnesium ions during electroelution, forming a stable spherical structure. At the same time, it neutralizes the negative charge on the DNA surface, causing it to stop migrating and remain stable in the sample recovery chamber.

[0016] Furthermore, in S2, the ultra-large DNA is a DNA fragment ranging in size from 100 kb to several Mb, including but not limited to bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), large genome fragments, and complete eukaryotic chromosomes, and is able to maintain the high integrity of the recovered DNA.

[0017] Furthermore, in S1, the biological sample is derived from prokaryotes or eukaryotes, including Escherichia coli, Saccharomyces cerevisiae, and mammalian cells; the total amount of ultra-large DNA obtained in a single recovery operation using this method exceeds 10 micrograms, and the recovered product can be directly used for downstream experimental applications such as transgenic and genome manipulation.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1) This method combines the excellent electrophoretic migration effect of pulsed-field gel electrophoresis for ultra-large DNA fragments with the principle of chain-globulin transformation. Pulsed-field gel electrophoresis is used to electrophores ultra-large DNA fragments from the gel block to the solution. In the PEG solution, the ultra-large DNA fragments are bound to magnesium ions and undergo chain-globulin transformation. The ultra-large DNA fragments electrophoresed are compressed and protected in real time. Electroelution and compression protection are integrated into a compact device to achieve efficient and complete in vitro purification and recovery of ultra-large DNA fragments. The ultra-large DNA fragments recovered by this method have extremely high integrity. 2) This invention is based on pulsed field gel electrophoresis for the efficient separation of ultra-large DNA fragments, and can recover a large amount of ultra-large DNA in one operation, up to 10 μg or more. 3) This method can separate, purify, and recover ultra-large DNA fragments ranging from hundreds of kb to several Mb; 4) This method is highly versatile and can recover very large DNA fragments from different species (such as bacteria, yeast, etc.); 5) The entire recycling operation is simple and fast. The use of a dedicated recycling device realizes the standardized process of "placement-electrophoresis-collection", which greatly reduces the operational difficulty of recycling ultra-large DNA fragments and shortens the recycling time. The entire recycling process for different sizes of target fragments only takes 1-12 hours. 6) The solvent for the ultra-large DNA products recovered by pulsed field gel electrophoresis contains only PEG polymers, and the recovered product has a single component, which facilitates further experimental operations on the recovered product. Attached Figure Description

[0019] Figure 1 Figure: Pulsed field gel electrophoresis elution and recovery device.

[0020] Wherein: A. Top view of the pulsed field gel electrophoresis elution and recovery device, where 1 is the electrophoresis protection chamber, 2 is the sample recovery chamber, solid lines represent the support material of the recovery device, dashed lines represent the semi-permeable membrane, dark blue squares represent gel blocks, and light blue squares represent removable and replaceable magnesium sheets; B. 3D schematic diagram of the pulsed field gel electrophoresis elution and recovery device, where 1 is the electrophoresis protection chamber, and 2 is the sample recovery chamber; C. Actual image of the pulsed field gel electrophoresis elution and recovery device, where 1 is the electrophoresis protection chamber, 2 is the sample recovery chamber, and 3 is the removable and replaceable magnesium sheet; D. Schematic diagram of the pulsed field gel electrophoresis elution and recovery device in operation, during recovery the device is placed in the center of the electrophoresis tank of the pulsed field gel electrophoresis apparatus.

[0021] Figure 2 The results show the elution and recovery of a large (pB, 456 Kb) human antibody gene cluster from E. coli by pulsed-field gel electrophoresis.

[0022] The process involved: A. Preparing *E. coli* containing the human antibody gene cluster fragment pB, which was eluted and recovered by pulsed-field gel electrophoresis (PGFGE), into PGFGE blocks. The size and concentration of the fragments were then determined by PGFGE. B. After eluting and recovering the human antibody gene cluster fragment pB by PGFGE, the recovered product was serially diluted and then subjected to PGFGE to determine its integrity and concentration. PGFGE conditions: 1 wt% agarose gel, 0.5*TBE buffer, 14... o C, change angle 120 o Voltage 6V / cm, pulse turning time 60s-120s, total electrophoresis time 24 h.

[0023] Figure 3 Results of the recovery of the spliced ​​GXE4 (1.14 Mb) genomic fragment of *Escherichia coli* from *Escherichia coli*.

[0024] The process involved: A. Preparing *E. coli* containing the *S. cristae* genome fragment GXE4 for pulsed-field gel electrophoresis (PGFGE) as PGFGE blocks, and then detecting the fragment size and concentration using PGFGE. B. After eluting and recovering the *S. cristae* genome fragment GXE4 using PGFGE, the recovered product was serially diluted and then analyzed for its integrity and concentration using PGFGE. PGFGE conditions: 1 wt% agarose gel, 0.5*TBE buffer, 14... o C, change angle 120 o Voltage 6V / cm, pulse turning time 60s-120s, total electrophoresis time 24 h.

[0025] Figure 4 Results of the recovery of chromosomes (0.2-2.2 Mb) from Saccharomyces cerevisiae BY4742.

[0026] The results included the recovery of the *Saccharomyces cerevisiae* BY4742 chromosome. A. After eluting and recovering *Saccharomyces cerevisiae* BY4742 into a pulsed-field gel electrophoresis (PGF) block, the fragment size and concentration were determined by PGF. B. After eluting and recovering the *Saccharomyces cerevisiae* BY4742 chromosome by PGF, the integrity and concentration of the recovered product were determined by PGF. PGF conditions: 1 wt% agarose gel, 0.5 × TBE buffer, 14... o C, change angle 120 o Voltage 6V / cm, pulse turning time 60s-120s, total electrophoresis time 24 h. Detailed Implementation

[0027] Overall, this invention aims to develop an in vitro electroelution recovery device and method that is simple to operate, highly efficient, rapid, and effectively maintains the integrity of ultra-large DNA fragments. Using this invention, ultra-large DNA fragments (bacterial artificial chromosomes, large genome fragments, etc.) ranging from hundreds of kb to several Mb can be rapidly separated and purified in vitro, with a total recovery volume exceeding 10 micrograms in a single operation. The purified and recovered ultra-large DNA exhibits high integrity and stability, and the storage solution for ultra-large DNA has a simple composition. Compared with other methods for recovering ultra-large DNA, this invention offers simple in vitro purification and recovery of ultra-large DNA, a short overall recovery time, a large total volume of ultra-large DNA fragments recovered in a single operation, high integrity, and long-term stable storage.

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Component models, material names, connection structures, circuit structures, control methods, algorithms, and other features not explicitly described in this technical solution are considered common technical features disclosed in the prior art.

[0029] Example 1 This invention provides an integrated in vitro electroelution and recovery device for ultra-large DNA samples. The device includes: 1. Electrophoresis protection room: as attached Figure 1 As shown in label 1 in A, B, and C, the electrophoresis protection chamber is a device with a hexagonal bottom. During electrophoresis, an electrophoresis protection solution is added to the electrophoresis protection chamber. The middle of its side is a mixed cellulose semipermeable membrane with a Millipore pore size of 0.025 μm. During pulsed field gel electrophoresis, the current can pass through the semipermeable membrane on the side and pass through the electrophoresis protection chamber.

[0030] 2. Sample recovery room: as attached Figure 1 As shown in label 2 of A, B, and C, this is a trapezoidal device with a semi-permeable membrane in the middle of its side. During electroelution recovery, the sample recovery chamber is placed in the middle of the electrophoresis protection chamber. The side of the sample recovery chamber is parallel to the side of the corresponding electrophoresis protection chamber, ensuring that the current during pulsed-field gel electrophoresis can pass through the semi-permeable membranes on the sides of both the electrophoresis protection chamber and the sample recovery chamber, traversing the entire recovery device. During electrophoresis, electrophoresis protection solution is added to the sample recovery chamber, and the sample to be recovered is placed in the middle of the semi-permeable membrane on the side of the sample recovery chamber.

[0031] 3. Removable and replaceable magnesium sheet: as shown in the attached document. Figure 1 As shown in mark 3 in C, the removable and replaceable magnesium sheet is placed in the electrophoresis protection chamber, parallel to the side of the sample recovery chamber and at a fixed distance, ranging from 1 to 2 cm. The magnesium sheet can be removed and replaced after each pulsed-field gel electrophoresis elution and recovery.

[0032] This invention provides a method for recovering ultra-large DNA fragments using the above-described apparatus.

[0033] The method includes the following steps: a. Sample preparation stage: After overnight culture and amplification of samples containing ultra-large DNA fragments (such as bacteria, yeast, etc.), pulsed field gel electrophoresis blocks are prepared with low melting point agarose at a certain concentration. After the gel blocks are prepared, they are digested with lysis buffer containing proteinase K, washed multiple times with TE buffer, and stored at 4°C for later use.

[0034] b. Electroelution Preparation Stage: Assemble the pulsed-field gel electrophoresis (PGF) device for recovering ultra-large DNA fragments. Place the PGF gel containing the target ultra-large DNA fragment in the sample recovery chamber, against the side semi-permeable membrane. Add recovery protection solution to both the sample recovery chamber and the electrophoresis protection chamber, ensuring the solution level just covers the gel fragment. Then, proceed with the entire recovery device as shown in the attached diagram. Figure 1 As shown in Figure D, the gel is placed in the electrophoresis tank of a pulsed field electrophoresis apparatus, and pulsed field gel electrophoresis buffer is added to the tank.

[0035] c. Electroelution stage: Power is turned on, and pulsed-field gel electrophoresis is performed under specific conditions (e.g., 14 °C, 6 V / cm, 60 s-120 s, 5 h). Under the influence of a periodically changing pulsed electric field, the large DNA molecules migrate out of the gel block and bind with magnesium ions ionized from the magnesium sheet. Under the action of the recovery protective solution, they are compressed into spheres. The negative charge on the surface of the large DNA molecules is also neutralized by the magnesium ions, thus stopping the migration of the large DNA molecules in the pulsed electric field. The large DNA molecules are collected in spherical form in the sample recovery chamber.

[0036] d. Sample collection: The solution containing the ultra-large DNA fragments, which was electrolyzed by pulsed field gel electrophoresis, was directly aspirated from the sample recovery chamber using a pipette. The recovered sample can be used directly for downstream experiments.

[0037] The core technology of this invention lies in using pulsed-field gel electrophoresis to drive ultra-large DNA molecules to migrate directionally from agarose gel blocks, achieving efficient purification and recovery through a specially designed device. The electrophoresis protection chamber provides a stable electric field environment, while the sample recovery chamber holds the DNA sample. A removable and replaceable magnesium plate ionizes to release magnesium ions that bind with the DNA to neutralize its charge. With the assistance of a recovery protection solution containing polyethylene glycol, the DNA molecules undergo compression transformation, forming a dense spherical structure, thereby stopping migration and accumulating in the recovery chamber. The entire process integrates separation, compression, and collection steps, overcoming the problems of product contamination and low efficiency in traditional methods, and achieving high-purity integrated recovery of ultra-large DNA molecules ranging from hundreds of kb to Mb.

[0038] Specifically, this invention innovatively integrates the molecular sieving effect of pulsed field gel electrophoresis with the metal ion-mediated biomacromolecule compression mechanism to achieve integrated purification and recovery of ultra-large DNA ranging from hundreds of kb to Mb. It drives the directional migration of DNA through a specific electric field configuration, while utilizing the charge neutralization effect of divalent magnesium ions on the DNA phosphate backbone, combined with the steric hindrance effect of polyethylene glycol, to induce a conformational transition of linear DNA to a compact spherical structure, thereby terminating electrophoretic migration and achieving in-situ aggregation. The electrophoresis protection chamber in the device forms a uniform electric field distribution through a polygonal container design and a semi-permeable membrane structure, providing a stable fluid environment for DNA migration. The detachable nature of the sample recovery chamber allows for flexible adaptation to different sample volumes. The placed magnesium sheet continuously ionizes magnesium ions under the electric field; these ions effectively shield electrostatic repulsion after binding with negatively charged DNA chains, reducing molecular spread. At the methodological level, the sample is first embedded in low-melting-point agarose and subjected to gentle lysis to maintain DNA integrity. Then, it is subjected to pulsed-field electrophoresis in a PEG-containing recovery protective solution. The alternating electric field direction causes the ultra-large DNA to continuously untangle from the gel matrix and migrate forward. When the DNA enters the magnesium ion-rich region, its interchain repulsion is weakened. PEG molecules further compress the DNA hydration layer by eliminating the volume effect, synergistically promoting the coil-globule phase transition of molecules to form nanoscale condensed particles. This compressed state not only inhibits the diffusion and migration of DNA in the electric field, but also significantly enhances its shear resistance. Finally, a high-concentration and high-purity recovery product can be obtained by simple liquid aspiration. The entire technology chain, through the precise coupling of physical field control, chemical equilibrium and biomolecular behavior, breaks through the bottlenecks of enzyme digestion residue, low recovery efficiency and lengthy operation in traditional methods, providing key technical support for genomics research and biotechnology applications.

[0039] Application Example 1 Pulsed-field gel electrophoresis was used to elute and recover the spliced ​​human antibody gene cluster fragment pB (456 Kb) from E. coli. In this example, the human antibody gene cluster fragment pB (456 Kb) that our research group had already assembled in E. coli was used as the pulsed-field gel electrophoresis elution sample to test the effectiveness of the pulsed-field gel electrophoresis elution recovery device and method in recovering ultra-large DNA fragments in the hundreds of Kb range.

[0040] (1) Sample preparation for pulsed-field gel electrophoresis elution: The *E. coli* containing the human antibody gene cluster fragment pB, to be recovered, was prepared into a pulsed-field gel electrophoresis block, and its concentration and integrity were detected by pulsed-field gel electrophoresis. The basic steps are as follows: 1. Pick a single E. coli clone containing the human antibody gene cluster fragment pB from the plate and transfer it to 50 mL LB medium. Incubate at 37°C. o C, incubate overnight at 220 rpm; 2. Take 250 μL of overnight cultured bacteria, dilute to 1 mL, and measure OD. λ=600nm Value, calculate the total concentration; 3. Collect the bacterial cells by centrifugation at 7000 rpm for 5 minutes; 4. Remove the supernatant, resuspend the E. coli in 25 mL TE25s solution (25 mM pH 8.0 Tris-HCl, 25 mM pH 8.0 EDTA, 10 wt% sucrose), and centrifuge at 7000 rpm for 5 minutes to collect the bacteria; 4. Calculate the total concentration of Escherichia coli and add the corresponding volume of TE25s solution to resuspend the Escherichia coli, so that each 50 μL of resuspension contains 50 D Escherichia coli; 5. Add an equal volume of 2wt% low-melting-point agarose (TE25s) and dissolve at 50°C. oAfter equilibration in a water bath (C), mix thoroughly by blowing and suction, then pour into a pulsed-field gel electrophoresis mold and freeze at 4°C. o C-cooling; 6. After 30 minutes, remove the gel block and add 10 mL of proteinase K lysis buffer (pH 8.0, 500 mM EDTA, 1 wt% sodium lauroyl sarcosinate, 200 μg / mL proteinase K) to each 1 mL gel block. o C digests for 12 hours; 7. Wash the gel block 4 times with 50 mL TE10 solution (10 mM pH 8.0 Tris-HCl, 10 mM pH 8.0 EDTA), and gently shake at room temperature for 30-60 minutes each time.

[0041] 8. Using the CHEF Mapper A7 pulsed-field gel electrophoresis system, 1 wt% agarose gel was applied in 0.5*TBE buffer for 14 hours. o Separation is performed under condition C, with the conversion angle set to 120°. o The pulse voltage was set to 6V / cm, the pulse electric field conversion time was 60s-120s, and the electrophoresis time was 24 hours.

[0042] The results of pulsed-field gel electrophoresis are attached. Figure 2 As shown in Figure A, E. coli containing the human antibody gene cluster fragment pB was digested with proteinase K lysis buffer in the gel block, exposing the ultra-large DNA fragment pB. After separation by pulsed-field gel electrophoresis, a high concentration and complete pB band were found. The sample gel block for elution and recovery by pulsed-field gel electrophoresis was ready.

[0043] (2) After the sample gel blocks are prepared, assemble the pulsed-field gel electrophoresis recovery device and prepare the pulsed-field gel electrophoresis buffer, etc., and start the pulsed-field gel electrophoresis elution and recovery of pB. The steps are as follows: The semi-permeable membrane is pasted in the middle of each side of the electrophoresis protection chamber and the sample recovery chamber. Magnesium sheets are cut and fixed in the electrophoresis protection chamber. The pasted sample recovery chamber is placed in the electrophoresis protection chamber and fixed. Prepare 2.5 L of 0.5*TBE buffer, take 50 mL and add 15 wt% PEG 20000, stir and mix thoroughly to dissolve PEG20000 to prepare electrophoresis recovery protection solution; Add the remaining 0.5*TBE buffer to the pulsed-field gel electrophoresis tank and turn on the circulation and cooling system to pre-cool the electrophoresis buffer to 14. o C; Place the prepared sample gel block against the semi-permeable membrane along the long side of the sample recovery chamber, and add the recovery protection solution to both the electrophoresis protection chamber and the sample recovery chamber so that the sample gel block is immersed in the protection solution; Start the pulsed-field gel electrophoresis program and set the recovery condition to a 120° angle transition.o Voltage 6V / cm, pulse turning time 60s, total electrophoresis time 10h; After pulsed-field gel electrophoresis and elution, the purified and compressed ultra-large DNA solution is directly aspirated from the sample recovery chamber using a pipette for downstream detection or experiments. The recovered ultra-large DNA solution was serially diluted and detected by pulsed-field gel electrophoresis using 1 wt% agarose gel in 0.5*TBE buffer for 14 hours. o Separation is performed under condition C. The conversion angle is set to 120°. o The voltage was set to 6V / cm, the pulse electric field conversion time was 60s-120s, and the electrophoresis time was 24 hours.

[0044] A portion of the recovered ultra-large DNA solution was used for Qubit quantification to determine the concentration of the recovered product.

[0045] The concentration of the recovered product from the ultra-large DNA fragment pB is shown in Table 1. The concentration of the recovered DNA was 4.27 ng / μl, the total volume of the recovered product was 7000 μl, and the total DNA recovery amount was 29.89 μg. The results of the integrity test of the recovered sample are attached. Figure 2 As shown in Figure B, the pB bands recovered by pulsed-field gel electrophoresis are clear, bright, and without tailing, indicating that the ultra-large DNA fragments recovered by pulsed-field gel electrophoresis have high integrity.

[0046] Table 1. Concentration of pB recovery product from human antibody gene cluster fragments quantified by Qubit. Application Example 2 Pulsed-field gel electrophoresis was used to elute and recover the spliced ​​*Stylosus crescentis* genome fragment GXE4 (1.14 Mb) from *E. coli*. Using the *Sterculia lunata* genome fragment GXE4 (1.14 Mb) assembled in *E. coli* as an example, the effectiveness of the pulsed-field gel electrophoresis (PLGE) elution and recovery device and method in recovering Mb-scale ultra-large DNA fragments was tested. The sample preparation procedure for PPLGE elution was the same as in Example 1, and the results of PPLGE detection of the eluted samples are attached. Figure 3 As shown in A. The pulsed-field gel electrophoresis elution procedure was basically the same as in Example 1, except that the pulse current switching time during pulsed-field gel electrophoresis elution was increased to 120 s. The pulsed-field gel electrophoresis recovery results of the *Stipa crescentis* genomic fragment GXE4 are shown in Table 2 and Appendix. Figure 3 As shown in Figure B. The concentration of recovered DNA was 1.89 ng / μl, the total volume of recovered product was 8000 μl, and the total recovered DNA amount was 15.12 μg. The results of the integrity test of the recovered sample are attached. Figure 3As shown in Figure B, the GXE4 band recovered by pulsed-field gel electrophoresis is clear, bright, and without tailing. These results indicate that high concentrations and intact Mb-scale ultra-large DNA fragments can be recovered in vitro via pulsed-field gel electrophoresis elution.

[0047] Table 2. Qubit Quantification of GXE4 Recovery Product Concentration of Bacillus cristae Genome Fragment GXE4 Application Example 3 Chromosomes of Saccharomyces cerevisiae BY4742 (0.2–2.2 Mb) were recovered by pulsed-field gel electrophoresis elution. In this example, the chromosome of Saccharomyces cerevisiae BY4742 (0.2-2.2 Mb) was used as the sample for pulsed-field gel electrophoresis elution to test the effectiveness of the pulsed-field gel electrophoresis elution recovery device and method in recovering ultra-large DNA fragments of different sizes in eukaryotic cells.

[0048] The basic steps for preparing sample blocks for pulsed-field gel electrophoresis elution and recovery using Saccharomyces cerevisiae BY4742 are as follows: 1. Pick activated Saccharomyces cerevisiae BY4742 single clones from the plate and inoculate them into YPAD medium. 30 o C, incubate overnight at 240 rpm; 2. Transfer the yeast cultured overnight in the test tube to 50 mL of YPAD medium, and initiate OD. λ=600nm =0.1; 3.30 o C, incubate at 240 rpm until OD λ=600nm =2.0, centrifuge at 5000 rpm for 5 minutes to collect bacteria; 4. Resuspend the yeast cells in 50 mL ddH2O, 20 o C. Centrifuge at 5000 rpm for 5 minutes to collect bacteria; 5. Resuspend yeast cells in 10 mL of pH 8.0, 10 mM EDTA, at 20°C. o C. Centrifuge at 5000 rpm for 5 minutes to collect bacteria; 6. Resuspend in 750 μL of pH 7.2, 10 mM Tris. HCl, transfer to a 1.5 mL EP tube, and incubate for 20 minutes. o C. Centrifuge at 5000 rpm for 5 minutes to collect bacteria; Resuspend in 150 μL of pH 7.2, 10 mM Tris. HCl, and place in a 50°C container. o Equilibrate in a water bath; 8. Add 150 μL of Zymolyse-20T solution (20 mg / mL Zymolyse-20T, 50 wt% glycerol, 2.5 wt% glucose, 50 mM pH 8.0 Tris.HCl) and 225 μL of 2 wt% TE25S dissolved low-melting-point agarose (prepared beforehand, at 50 mM pH 8.0 Tris.HCl). o (Balance in a water bath at room temperature), mix well, pour into a mold, and refrigerate at room temperature. o C-cooling; 9. After 30 minutes, remove the gel block and add 5 mL of lysis buffer (pH 7.5, 10 mM Tris. HCl and pH 8.0, 50 mM EDTA) and 500 μL of Zymolyse-20T at 37°C. o Incubate for 3 hours; 10. Wash the gel block once with 25 mL ddH2O, then wash once with washing buffer (pH 7.5, 20 mM Tris.HCl and pH 8.0, 50 mM EDTA); 11. Add 5 mL of protease reaction solution (pH 8.0, 100 mM EDTA, 0.2 wt% acetylcholine, 1 wt% sodium lauroyl sarcosinate, 1 mg / mL proteinase K) to each 1 mL gel block. o C digests for 48 hours; 12. Wash the gel block four times with 50 mL of wash buffer, gently shaking at room temperature for 30-60 minutes each time; 13. Using the CHEF Mapper A7 pulsed-field gel electrophoresis system, 1 wt% agarose gel was applied in 0.5*TBE buffer for 14 minutes. o Separation is performed under condition C. The conversion angle is set to 120°. o The voltage was set to 6V / cm, the pulse electric field conversion time was 60s-120s, and the electrophoresis time was 24 hours.

[0049] The results of pulsed-field gel electrophoresis are attached. Figure 4 As shown in Figure A, the cell walls and intracellular proteins of Saccharomyces cerevisiae BY4742 were digested with lysis buffer within the gel block, exposing large DNA fragments of yeast chromosomes. After separation by pulsed-field gel electrophoresis, high concentrations of intact yeast chromosome fragments of different sizes were obtained. The sample gel block for elution and recovery by pulsed-field gel electrophoresis was ready.

[0050] The pulsed-field gel electrophoresis (PGF) elution procedure for recovering the *Saccharomyces cerevisiae* BY4742 chromosome was basically the same as in Examples 1 and 2, except that the pulse electric field switching time during PGF elution was changed to 60-120 s, and the elution and recovery time was 12 h. The results of the PGF elution recovery of *Saccharomyces cerevisiae* BY4742 chromosome are shown in Table 3 and Appendix. Figure 4 As shown in Figure B. The concentration of recovered DNA was 3.47 ng / μl, the total volume of recovered product was 7000 μl, and the total recovered DNA amount was 24.27 μg. The results of the integrity test of the recovered sample are attached. Figure 4 As shown in Figure B, the Saccharomyces cerevisiae chromosome bands recovered by pulsed-field gel electrophoresis were clear, bright, and without tailing. These results indicate that pulsed-field gel electrophoresis can recover high concentrations and intact, large DNA fragments of eukaryotic chromosomes.

[0051] Table 3. Concentration of chromosome recovery products from Saccharomyces cerevisiae BY4742 quantified by Qubit. The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A device for eluting, purifying, and recovering ultra-large DNA based on pulsed-field gel electrophoresis, characterized in that, include: The electrophoresis protection chamber (1) is a polygonal container structure with a semi-permeable membrane on its side. The electrophoresis protection chamber (1) is used to contain the electrophoresis protection solution and allow the current to pass through the semi-permeable membrane during the pulsed field gel electrophoresis process. The sample recovery chamber (2) is a polygonal container structure with a semi-permeable membrane in the middle of its side. The sample recovery chamber (2) is detachably placed in the center of the electrophoresis protection chamber (1), and its side is parallel to the corresponding side of the electrophoresis protection chamber, so that the current passes through evenly. The sample recovery chamber (2) is used to place the ultra-large DNA sample gel block to be recovered and add electrophoresis protection solution. A removable and replaceable magnesium sheet (3) is provided in the electrophoresis protection chamber (1). The removable and replaceable magnesium sheet (3) is parallel to the side of the sample recovery chamber and maintains a predetermined distance. It is used to ionize magnesium ions during electrophoresis to promote the compression and protection of ultra-large DNA.

2. The apparatus for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution according to claim 1, characterized in that, The semi-permeable membranes of the electrophoresis protection chamber (1) and the sample recovery chamber (2) are made of materials that allow ions and small molecules to pass through but block the passage of ultra-large DNA, so that ultra-large DNA is retained in the sample recovery chamber (2) during the electroelution process.

3. The apparatus for eluting, purifying, and recovering ultra-large DNA based on pulsed-field gel electrophoresis according to claim 1, characterized in that, The electrophoresis protection chamber (1) is a container with a hexagonal bottom surface, and the sample recovery chamber (2) is a container with a trapezoidal bottom surface; The electrophoresis protection chamber (1), the sample recovery chamber (2), and the detachable and replaceable magnesium sheet (3) are all independent components. When using them, the components are assembled and then the ultra-large DNA samples are recovered. The electrophoresis protection chamber (1) and sample recovery chamber (2) are soaked in ultrapure water for long-term storage after use, and can be reused multiple times.

4. A method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution, characterized in that, Includes the following steps: S1. After culturing and concentrating biological samples containing ultra-large DNA fragments, pulsed field gel electrophoresis blocks were prepared using low-melting-point agarose. The gel blocks were then subjected to lysis and washing to expose the ultra-large DNA. S2. Assemble the device as described in any one of claims 1 to 3, place the treated gel block in the sample recovery chamber (2) and place it close to the side semi-permeable membrane, add a recovery protection solution containing PEG polymer to the sample recovery chamber (2) and the electrophoresis protection chamber (1) so that the liquid surface submerges the pulsed field gel electrophoresis gel block, and then place the entire device in the pulsed field gel electrophoresis tank and add electrophoresis buffer. S3. Electrophoresis is performed under pulsed field gel electrophoresis conditions, which allows the super-large DNA to migrate out of the gel block and combine with the magnesium ions ionized by the removable and replaceable magnesium sheet (3). Under the action of the recovery protective solution, the DNA is compressed into a spherical shape, thereby neutralizing the charge and stopping the migration, and collected in the sample recovery chamber. S4. Take the solution containing compressed super-large DNA from the sample recovery chamber (2).

5. The method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution according to claim 4, characterized in that, In S1, the lysis process involves digesting the gel block at 50°C for 12 to 48 hours using a lysis buffer containing proteinase K, wherein the lysis buffer contains 500 mM EDTA at pH 8.0, 1 wt% sodium lauroyl sarcosinate and 200 μg / mL proteinase K. The cleaning process involves washing the digested gel block multiple times at room temperature using a pH 8.0 TE buffer solution, with each wash lasting 30 to 60 minutes.

6. The method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution according to claim 4, characterized in that, In S3, the pulsed field gel electrophoresis conditions specifically include: electrophoresis temperature controlled at 7-14℃, electric field strength set at 2-6V / cm, pulsed electric field conversion time of 30 seconds to 500 seconds, pulsed electric field conversion angle of 106°-120°, and total electrophoresis time adjusted between 1 hour and 12 hours according to the size of the target DNA fragment, so that ultra-large DNA fragments can migrate out of the gel block and undergo strand-globulin conversion.

7. The method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution according to claim 6, characterized in that, In S3, the pulsed electric field switching time is specifically adjusted according to the size of the ultra-large DNA fragment to be recovered, specifically as follows: When the recovered DNA fragments are in the hundreds of kb range, the pulse turn-off time is set to 30-90 seconds; When the recovered DNA fragments are in the Mb range, the pulse turn-off time is set to 90-500 seconds to accommodate the migration characteristics of DNA fragments of different sizes in the pulse field, thereby optimizing separation efficiency and recovery yield.

8. The method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution according to claim 4, characterized in that, In S2, the recovered protective liquid contains 15 wt% PEG polymer with a degree of polymerization ranging from 3000 to 20000. The PEG polymer is dissolved in 0.5×TBE buffer. The PEG polymer is used to promote the chain-to-sphere transformation of super-large DNA after binding with magnesium ions during electroelution, forming a stable spherical structure. At the same time, it neutralizes the negative charge on the DNA surface, causing it to stop migrating and remain stable in the sample recovery chamber.

9. The method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution according to claim 6, characterized in that, In S2, the ultra-large DNA is a DNA fragment with a size ranging from 100 kb to several Mb.

10. The method for purifying and recovering ultra-large DNA based on pulsed-field gel electrophoresis elution according to claim 6, characterized in that, In S1, the biological sample is derived from prokaryotes or eukaryotes, including Escherichia coli, Saccharomyces cerevisiae, or mammalian cells.

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