A recombinant topoisomerase and its use in plasmid linearization
By recombining topoisomerase I with T7 endonuclease I, the problem of linearizing unknown sequence plasmids was solved, achieving efficient linearization and full-length sequencing of unknown plasmids.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING TRANSGEN BIOTECH CO LTD
- Filing Date
- 2026-02-11
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies struggle to achieve efficient linearization on plasmids with unknown sequences, and restriction endonuclease methods rely on known sequences, making them unsuitable for third-generation sequencing technologies.
By recombinantly modifying topoisomerase I to remove its ligation activity and retain only its activity in cleaving supercoiled plasmids, and then combining it with T7 endonuclease I for cleavage, the plasmids were linearized.
It achieves efficient linearization of plasmids with unknown sequences, is applicable to third-generation sequencing technology, and provides a fast and widely applicable plasmid linearization method.
Smart Images
Figure CN121874170B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology. More specifically, it relates to a recombinant topoisomerase and its application in plasmid linearization. Background Technology
[0002] Plasmid linearization is the process of converting circular plasmids into linear molecules, commonly used in experiments such as PCR, in vitro transcription, and the construction of stable cell lines. Restriction endonucleases (REs) are enzymes that recognize specific DNA sequences and cut them at specific sites within those sequences. Enzymatic digestion using restriction endonucleases is a common method for plasmid linearization, but this method requires the plasmid sequence to be known.
[0003] Third-generation sequencing (TGS) offers advantages such as extremely long read lengths, no PCR amplification required, no GC bias, and short sequencing cycles, making it suitable for complex genome assembly, epigenetic research, and large-fragment variation detection. TGS enables full-length plasmid sequencing, allowing for rapid and accurate detection of backbone deletions, fragment recombination, and other issues. Since most plasmids are circular molecules, plasmid linearization is necessary before constructing a TGS library. However, for plasmids with completely unknown sequences, it is difficult to select suitable restriction endonucleases to perform the linearization process.
[0004] Topoisomerases catalyze the cleavage and recombination of DNA strands, thereby altering the topological conformation of DNA. Based on their mechanism of action, topoisomerases can be divided into two classes: topoisomerase I and topoisomerase II. Topoisomerase I catalyzes the cleavage and recombination of one strand of a DNA double helix, and this enzyme is not strictly dependent on recognizing a specific DNA sequence.
[0005] Therefore, a novel plasmid linearization method can be developed. By recombinantly modifying topoisomerase I to retain only its cleavage activity on supercoiled plasmids, an open-circular plasmid product can be obtained. Then, T7 endonuclease I (T7EI) is used to cleave the cleavage site to finally obtain a linearized product, which can be applied to third-generation sequencing of plasmids with unknown sequences. Summary of the Invention
[0006] One object of the present invention is to provide a recombinant topoisomerase, which, by recombinantly modifying topoisomerase I to remove its ligation activity and retain only its cleavage activity on supercoiled plasmids, can yield open-ring plasmid products.
[0007] Another objective of this invention is to provide the application of the above-mentioned recombinant topoisomerase in plasmid linearization. When used in conjunction with T7 endonuclease I (T7EI), it can linearize plasmids with different initial input amounts and fragment sizes under the premise of unknown sequence. Combined with third-generation sequencing technology, it can realize full-length sequencing of unknown plasmids.
[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a recombinant topoisomerase, the amino acid sequence of which is shown in SEQ ID NO.1.
[0009] In a second aspect, the present invention provides an enzyme composition comprising the above-mentioned recombinant topoisomerase and T7 endonuclease I.
[0010] The recombinant topoisomerase of this invention is a multi-site mutation of Escherichia coli type I topoisomerase (amino acid sequence as shown in SEQ ID NO.2), which removes the ligation activity of the enzyme, allowing it to form cleavage sites on supercoiled plasmid substrates to obtain open-ring plasmid products; then, T7 endonuclease I is used to cut the cleavage sites to achieve plasmid linearization.
[0011] Thirdly, the present invention provides the use of the above-described recombinant topoisomerase and / or enzyme composition in plasmid linearization or in the preparation of plasmid linearized products.
[0012] Fourthly, the present invention provides a plasmid linearization reaction solution, comprising a linearization enzyme mixture and a linearization reaction buffer; The linearized enzyme mixture includes the aforementioned recombinant topoisomerase and T7 endonuclease I.
[0013] In a specific implementation, the T7 endonuclease I was purchased from TransGen, catalog number LE101.
[0014] In a specific embodiment, the final concentration of the recombinant topoisomerase in the linearized enzyme mixture is 20-50 ng / μL; the final concentration of the T7 endonuclease I in the linearized enzyme mixture is 0.1-0.5 U / μL.
[0015] In a preferred embodiment, the final concentration of the recombinant topoisomerase in the linearized enzyme mixture is 40 ng / μL; and the final concentration of the T7 endonuclease I in the linearized enzyme mixture is 0.2 U / μL.
[0016] In a specific embodiment, the linearizing enzyme mixture further includes Tris-HCl at pH 8.0, (NH4)2SO4, KCl, DTT, Tween 20, and glycerol; wherein the final concentration of Tris-HCl at pH 8.0 in the linearizing enzyme mixture is 40-60 mM; the final concentration of (NH4)2SO4 in the linearizing enzyme mixture is 20-40 mM; the final concentration of KCl in the linearizing enzyme mixture is 40-60 mM; the final concentration of DTT in the linearizing enzyme mixture is 0.5-5 mM; the final concentration of Tween 20 in the linearizing enzyme mixture is 0.05-0.2% (V / V); and the final concentration of glycerol in the linearizing enzyme mixture is 40-60% (V / V).
[0017] In a preferred embodiment, the final concentration of Tris-HCl at pH 8.0 in the linearized enzyme mixture is 50 mM; the final concentration of (NH4)2SO4 in the linearized enzyme mixture is 30 mM; the final concentration of KCl in the linearized enzyme mixture is 50 mM; the final concentration of DTT in the linearized enzyme mixture is 1 mM; the final concentration of Tween 20 in the linearized enzyme mixture is 0.1% (V / V); and the final concentration of glycerol in the linearized enzyme mixture is 50% (V / V).
[0018] In a specific embodiment, the linearization reaction buffer includes a pH buffer medium, a magnesium salt, a monovalent cation salt, and a reducing agent.
[0019] In a preferred embodiment, the pH buffer medium comprises either Tris-HCl or Tris-acetic acid at pH 8.0; the magnesium salt comprises either MgCl2 or MgAc2; the monovalent cation salt comprises either NaCl or KCl; and the reducing agent comprises either DTT or TCEP.
[0020] In a preferred embodiment, the linearization reaction buffer consists of Tris-HCl, MgCl2, KCl, and DTT at pH 8.0.
[0021] In a preferred embodiment, the final concentration of Tris-HCl at pH 8.0 in the linearization reaction buffer is 50-200 mM; the final concentration of MgCl2 in the linearization reaction buffer is 25-60 mM; the final concentration of KCl in the linearization reaction buffer is 100-500 mM; and the final concentration of DTT in the linearization reaction buffer is 25-75 mM.
[0022] In a preferred embodiment, the final concentration of Tris-HCl at pH 8.0 in the linearization reaction buffer is 100 mM; the final concentration of MgCl2 in the linearization reaction buffer is 50 mM; the final concentration of KCl in the linearization reaction buffer is 250 mM; and the final concentration of DTT in the linearization reaction buffer is 50 mM.
[0023] Fifthly, a plasmid linearization kit is provided, comprising the above-mentioned plasmid linearization reaction solution.
[0024] In a specific embodiment, the plasmid linearization kit further includes a reaction termination solution.
[0025] In a specific embodiment, the reaction termination solution includes EDTA.
[0026] In a preferred embodiment, the final concentration of EDTA in the reaction termination solution is 50 to 200 mM.
[0027] In a preferred embodiment, the final concentration of EDTA in the reaction termination solution is 100 mM.
[0028] Sixthly, a method for plasmid linearization reaction is provided, wherein the plasmid is linearized using the above-mentioned plasmid linearization reaction solution, a reaction termination solution is added, and the plasmid is purified.
[0029] In a specific implementation, the linearization reaction is carried out at a temperature of 50 to 75°C for a time of 5 to 15 minutes.
[0030] In a preferred embodiment, the linearization reaction is carried out at a temperature of 70°C for 10 minutes.
[0031] In a specific implementation, the purification is performed using DNA sorting magnetic beads. MagicPure ® Size Selection DNA Beads (TransGen, EC401) were purified.
[0032] In a specific implementation, the amount of plasmid added is 50 ng to 2 μg.
[0033] The beneficial effects of this invention are as follows: The recombinant topoisomerase of this invention does not depend on recognizing a specific DNA sequence. It can form cleavage sites on supercoiled plasmids, and then obtain linear products through cleavage by T7 endonuclease I, thus achieving the linearization of plasmids with unknown sequences. The plasmid linearization kit developed using the recombinant topoisomerase of this invention is rapid, efficient, and widely applicable. Combined with third-generation sequencing technology, it can achieve full-length sequencing of unknown plasmids. Attached Figure Description
[0034] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
[0035] Figure 1 The results are agarose gel electrophoresis of the linearization reaction of the 10,491 bp plasmid using different linearization reaction solutions. Figure 2 The results are agarose gel electrophoresis of the linearization reaction of 10,491 bp plasmids with different initial input amounts using linearization reaction solution 8. Figure 3 The results are obtained by agarose gel electrophoresis of 200 ng plasmids of different fragment sizes using linearization reaction solution 8. Detailed Implementation
[0036] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.
[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The sources of some reagents in the examples are as follows: T7 endonuclease I: TransGen, catalog number LE101; DNA sorting magnetic beads: MagicPure ® Size Selection DNA Beads (TransGen, catalog number EC401); Restriction endonucleases: XhoI rapid endonuclease (TransGen, catalog number JX201), NotI rapid endonuclease (TransGen, catalog number JN401), BamHI rapid endonuclease (TransGen, catalog number JB101), NcoI rapid endonuclease (TransGen, catalog number JN101), XbaI rapid endonuclease (TransGen, catalog number JX101); 15K DNA molecular weight standard (i.e., marker): TransGen, catalog number BM161; Third-generation sequencing library preparation kits: Amplification-free barcode sequencing kit (ONT, SQK-NBD114.96) and transposase-based rapid barcode sequencing kit (ONT, SQK-RBK114.96).
[0038] Example 1 Construction of recombinant topoisomerase Multi-site mutations were performed on Escherichia coli type I topoisomerase (amino acid sequence shown in SEQ ID NO.2), and recombinant topoisomerase with delinking activity was obtained through screening. This recombinant topoisomerase can form cleavage sites on supercoiled plasmid substrates, yielding open-ring plasmid products. Further linearization can be obtained by combining it with T7 endonuclease I.
[0039] Finally, the amino acid sequence of the recombinant topoisomerase is shown in SEQ ID NO.1.
[0040] Example 2: Plasmid linearization reaction using recombinant topoisomerase and T7 endonuclease I I. Preparation of plasmid linearization reaction solution and reaction termination solution 1) Preparation of plasmid linearization reaction solution The plasmid linearization reaction solution consists of two parts: a linearizing enzyme mixture and a linearization reaction buffer. Different linearization reaction solutions can be obtained by combining different linearizing enzyme mixtures and linearization reaction buffers.
[0041] 1. Preparation of linearized enzyme mixture The components of linearizing enzyme mixture I and their final concentrations in linearizing enzyme mixture I are as follows: recombinant topoisomerase (10 ng / μL), T7 endonuclease I (0.1 U / μL), Tris-HCl at pH 8.0 (50 mM), (NH4)2SO4 (30 mM), KCl (50 mM), DTT (1 mM), Tween 20 (V / V, 0.1%), and glycerol (V / V, 50%).
[0042] The components of linearizing enzyme mixture II and their final concentrations in linearizing enzyme mixture II are as follows: recombinant topoisomerase (20 ng / μL), T7 endonuclease I (0.1 U / μL), Tris-HCl at pH 8.0 (50 mM), (NH4)2SO4 (30 mM), KCl (50 mM), DTT (1 mM), Tween 20 (V / V, 0.1%), and glycerol (V / V, 50%).
[0043] The components of linearizing enzyme mixture III and their final concentrations in linearizing enzyme mixture III are as follows: recombinant topoisomerase (40 ng / μL), T7 endonuclease I (0.2 U / μL), Tris-HCl at pH 8.0 (50 mM), (NH4)2SO4 (30 mM), KCl (50 mM), DTT (1 mM), Tween 20 (V / V, 0.1%), and glycerol (V / V, 50%).
[0044] The components of linearizing enzyme mixture IV and their final concentrations in linearizing enzyme mixture IV are as follows: recombinant topoisomerase (50 ng / μL), T7 endonuclease I (0.5 U / μL), Tris-HCl at pH 8.0 (50 mM), (NH4)2SO4 (30 mM), KCl (50 mM), DTT (1 mM), Tween 20 (V / V, 0.1%), and glycerol (V / V, 50%).
[0045] The components of linearizing enzyme mixture V and their final concentrations in linearizing enzyme mixture V are as follows: recombinant topoisomerase (50 ng / μL), T7 endonuclease I (1 U / μL), Tris-HCl at pH 8.0 (50 mM), (NH4)2SO4 (30 mM), KCl (50 mM), DTT (1 mM), Tween 20 (V / V, 0.1%), and glycerol (V / V, 50%).
[0046] 2. Preparation of linearization reaction buffer The components of linearization reaction buffer I and the final concentrations of each component in linearization reaction buffer I: Tris-acetic acid (100 mM), MgAc2 (50 mM), NaCl (250 mM), and TCEP (5 mM) at pH 8.0.
[0047] The components of linearization reaction buffer II and their final concentrations in linearization reaction buffer II: Tris-HCl (100 mM), MgCl2 (50 mM), KCl (250 mM), and DTT (50 mM) at pH 8.0.
[0048] 3. Combination of linearized reaction solutions Linearization reaction solution 1: Linearization enzyme mixture I + Linearization reaction buffer I; Linearization reaction solution 2: Linearization enzyme mixture II + Linearization reaction buffer I; Linearization reaction solution 3: Linearization enzyme mixture III + Linearization reaction buffer I; Linearization reaction solution 4: Linearization enzyme mixture IV + Linearization reaction buffer I; Linearization reaction solution 5: Linearization enzyme mixture V + Linearization reaction buffer I; Linearization reaction solution 6: Linearization enzyme mixture I + Linearization reaction buffer II; Linearization reaction solution 7: Linearization enzyme mixture II + Linearization reaction buffer II; Linearization reaction solution 8: Linearization enzyme mixture III + Linearization reaction buffer II; Linearization reaction solution 9: Linearization enzyme mixture IV + Linearization reaction buffer II; Linearization reaction solution 10: Linearization enzyme mixture V + Linearization reaction buffer II.
[0049] II. Preparation of the reaction termination solution The reaction termination solution consisted of 100 mM EDTA.
[0050] II. Linearization reaction of plasmids Linearization reactions were carried out using plasmids of different starting amounts and fragment sizes as substrates and different linearization reaction solutions. The reaction systems are shown in Table 1. The linearization reaction conditions were: incubation at 70℃ for 10 min, followed by the addition of 2 μL of reaction stop solution after the reaction, and the use of 12 μL (1×) DNA sorting magnetic beads. MagicPure ® The product was purified using size selection DNA beads (TransGen, EC401).
[0051] Table 1 Linearized reaction system
[0052] Based on the plasmid sequence, a restriction endonuclease with only one recognition site was selected to linearize the plasmid as a positive control. The reaction system is shown in Table 2. The linearization reaction conditions were: incubation at 37℃ for 10 min followed by incubation at 65℃ for 20 min. After the reaction, 20 μL (1×) DNA sorting magnetic beads were used. MagicPure ® The product was purified using size selection DNA beads (TransGen, EC401). Product yield and length distribution were determined by Qubit and 0.8% agarose gel electrophoresis, respectively.
[0053] Table 2 Enzyme digestion reaction system for restriction endonucleases
[0054] Specifically as follows: 1. Linearization effect of different linearization reaction solutions Using 200 ng of a 10,491 bp plasmid as substrate, linearization reactions were performed using different linearization reaction solutions and NotI restriction endonucleases. The results are shown in Table 3 and [Table data missing]. Figure 1 As shown.
[0055] Table 3. Yields of linearized products obtained using different linearization reaction solutions
[0056] 2. Linearization effect of plasmids with different initial input amounts Using a 10,491 bp plasmid as substrate, initial input amounts of 2 μg, 1.5 μg, 1 μg, 800 ng, 500 ng, 200 ng, 100 ng, and 50 ng were used for linearization reactions with linearization reaction solution 8 and NotI restriction endonuclease, respectively. The results are shown in Table 4. Figure 2 As shown.
[0057] Table 4. Yields of linearized products from plasmids with different initial input amounts
[0058] 3. Linearization effect of plasmids with different fragment sizes Using 200 ng of plasmid as substrate, plasmid fragments of 4,629 bp, 10,491 bp, 17,977 bp, 20,577 bp, and 31,797 bp were linearized using linearization reaction solution 8 and different restriction endonucleases. The results are shown in Table 5. Figure 3 As shown.
[0059] Table 5. Yields of linearized products from plasmids of different fragment sizes
[0060] Analysis of the above results shows that the sizes of the products obtained by linearizing plasmids using linearization reaction solutions 2, 3, 4, 7, 8, and 9 are basically consistent with those of the positive control group using restriction endonucleases. This indicates that when the final concentration of recombinant topoisomerase in the linearization enzyme mixture is 20-50 ng / μL and the final concentration of T7 endonuclease I is 0.1-0.5 U / μL, this enzyme combination can achieve a good linearization effect. The yield of products obtained by linearizing plasmids using linearization reaction solutions 7-9 (i.e., combined with linearization reaction buffer II) is relatively higher, indicating that linearization reaction buffer II (pH 8.0 Tris-HCl (100 mM), MgCl2 (50 mM), KCl (250 mM), DTT (50 mM)) is determined to be a better linearization reaction buffer, which can obtain a higher yield of linearized products. Linearization reaction solution 8 is effective for different initial input amounts (50 ng-2 μg) and different fragment sizes (4,629-2 μg). The plasmid (31,797 bp) was linearized, and the yield was comparable to that of the corresponding restriction endonuclease control group. This indicates that when the enzyme composition is 40 ng / μL recombinant topoisomerase and 0.2 U / μL T7 endonuclease I, and is used in conjunction with linearization reaction buffer II (pH 8.0 Tris-HCl (100 mM), MgCl2 (50 mM), KCl (250 mM), DTT (50 mM)), linearization of plasmids with different starting amounts and fragment sizes can be achieved.
[0061] Example 3: Terminal sequence analysis of plasmid linearization products In this embodiment, plasmids of different fragment sizes were used as substrates, with fragment sizes of 4,629 bp, 10,491 bp, 17,977 bp, 20,577 bp, and 31,797 bp, respectively. Linearization reactions were performed using linearization reaction solution 8 and different restriction endonucleases. Then, a third-generation sequencing library was constructed using an amplification-free barcode sequencing kit (ONT, SQK-NBD114.96), and the libraries were sent to a third-party company for sequencing and analysis. After removing adapters and barcode sequences, the composition of the last 5 bases of the sequence was statistically analyzed, and the results are shown in Table 6.
[0062] Table 6. Base composition at the ends of plasmid sequences
[0063] Analysis of the above results shows that the recognition sequences of restriction endonucleases are relatively fixed, so the plasmid sequence must be known in order to select the appropriate restriction endonuclease for linearization; while the linearization system using recombinant topoisomerase does not depend on recognizing a specific DNA sequence, so it can achieve linearization of plasmids with unknown sequences.
[0064] Example 4: N50 data analysis of third-generation plasmid sequencing libraries In this example, plasmids of different fragment sizes were used as substrates, with fragment sizes of 4,629 bp, 10,491 bp, 17,977 bp, 20,577 bp, and 31,797 bp, respectively. Third-generation sequencing libraries were constructed using a transposase-based rapid barcoding sequencing kit (ONT, SQK-RBK114.96), and then sent to a third-party company for sequencing and analysis. The N50 data of the third-generation sequencing libraries constructed using linearization reaction solution 8 and an amplification-free barcoding sequencing kit (ONT, SQK-NBD114.96) in Example 3 were compared and analyzed. The results are shown in Table 7.
[0065] Table 7. N50 data analysis of third-generation sequencing libraries constructed using different kits.
[0066] Analysis of the above results shows that while transposase-based rapid barcode sequencing kits can convert circular plasmids into linear plasmids, they also further fragment the linear plasmids into smaller fragments. In contrast, linearization systems using recombinant topoisomerases can yield plasmid fragments that are close to full length, which is helpful for assembling the original sequences of unknown plasmids.
[0067] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.
Claims
1. A recombinant topoisomerase, characterized in that, The amino acid sequence of the recombinant topoisomerase is shown in SEQ ID NO.
1.
2. An enzyme composition, characterized in that, The enzyme composition comprises the recombinant topoisomerase of claim 1 and T7 endonuclease I; when used, the enzyme composition is prepared as a linearized enzyme mixture, wherein the final concentration of the recombinant topoisomerase in the linearized enzyme mixture is 20-50 ng / μL; and the final concentration of the T7 endonuclease I in the linearized enzyme mixture is 0.1-0.5 U / μL.
3. The use of the recombinant topoisomerase of claim 1 or the enzyme composition of claim 2 in plasmid linearization or in the preparation of plasmid linearized products.
4. A plasmid linearization reaction solution, characterized in that, The plasmid linearization reaction solution includes a linearization enzyme mixture and a linearization reaction buffer; wherein the linearization enzyme mixture includes the enzyme composition according to claim 2.
5. The plasmid linearization reaction solution according to claim 4, characterized in that, The linearizing enzyme mixture further includes Tris-HCl at pH 8.0, (NH4)2SO4, KCl, DTT, Tween 20, and glycerol; wherein the final concentration of Tris-HCl at pH 8.0 in the linearizing enzyme mixture is 40-60 mM; the final concentration of (NH4)2SO4 in the linearizing enzyme mixture is 20-40 mM; the final concentration of KCl in the linearizing enzyme mixture is 40-60 mM; the final concentration of DTT in the linearizing enzyme mixture is 0.5-5 mM; the final concentration of Tween 20 in the linearizing enzyme mixture is 0.05-0.2%; and the final concentration of glycerol in the linearizing enzyme mixture is 40-60%.
6. The plasmid linearization reaction solution according to claim 4, characterized in that, The linearization reaction buffer solution includes a pH buffer medium, a magnesium salt, a monovalent cation salt, and a reducing agent.
7. The plasmid linearization reaction solution according to claim 6, characterized in that, The pH buffer medium includes either Tris-HCl or Tris-acetic acid at pH 8.0; the magnesium salt includes either MgCl2 or MgAc2; the monovalent cation salt includes either NaCl or KCl; and the reducing agent includes either DTT or TCEP.
8. The plasmid linearization reaction solution according to claim 7, characterized in that, The linearization reaction buffer consists of Tris-HCl, MgCl2, KCl and DTT at pH 8.
0.
9. The plasmid linearization reaction solution according to claim 8, characterized in that, The final concentration of Tris-HCl at pH 8.0 in the linearization reaction buffer is 50-200 mM; the final concentration of MgCl2 in the linearization reaction buffer is 25-60 mM; the final concentration of KCl in the linearization reaction buffer is 100-500 mM; and the final concentration of DTT in the linearization reaction buffer is 25-75 mM.
10. A plasmid linearization kit, characterized in that, The plasmid linearization kit includes the plasmid linearization reaction solution according to any one of claims 4-9.
11. The plasmid linearization kit according to claim 10, characterized in that, The plasmid linearization kit also includes a reaction termination solution.
12. A method for plasmid linearization reaction, characterized in that, The method uses the plasmid linearization reaction solution according to any one of claims 4-9 to linearize the plasmid, adds the reaction termination solution, and purifies it.
13. The method according to claim 12, characterized in that, The linearization reaction is carried out at a temperature of 50 to 75°C for a time of 5 to 15 minutes.