A ligase buffer and its products and their application in mRNA tailing

Abstract

This invention provides a ligase buffer and its product, and its application in mRNA tailing, relating to the field of nucleic acid synthesis technology. The invention provides a T4 DNA ligase buffer comprising Tris-HCl, ATP, MgCl2, reduced glutathione (GSH), polyvinylpyrrolidone K30 (PVP K30), and Triton X-100; this buffer significantly improves the ligation efficiency of T4 DNA ligase and can be applied to mRNA tailing based on enzyme digestion and ligation.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid synthesis technology, and relates to DNA ligation, specifically to a ligase buffer and its products and their application in mRNA tailing. Background Technology

[0002] In molecular biology experiments, gene cloning, DNA library construction, and gene editing, DNA ligases are key enzymes that catalyze the formation of phosphodiester bonds at the ends of DNA fragments. The integrity of their function directly determines the success or failure of experiments. DNA ligases specifically catalyze the covalent linkage between DNA fragments with 5'-phosphate groups and 3'-hydroxyl ends, restoring the continuity of the DNA strand. They are indispensable "molecular glue" in recombinant DNA technology. Based on differences in coenzyme type and source, DNA ligases are mainly divided into ATP-dependent and NAD-dependent types. + There are two main categories of ATP-dependent ligases. T4 DNA ligase, a typical representative of ATP-dependent ligases, is widely used in routine molecular cloning experiments due to its ability to simultaneously ligate sticky-end and blunt-end DNA fragments and its strong compatibility with modified bases and various double-stranded DNA structures. Thermostable DNA ligases (such as Tba ligase and Taq ligase derived from hyperthermophilic archaea) exhibit unique advantages in ligation reactions that rely on high temperatures for enhanced specificity due to their high-temperature activity stability, but their natural fidelity and substrate adaptability remain limited. Thermostable ligases (such as T4 DNA ligase), while widely used, suffer from inherent poor thermostability. They are prone to irreversible denaturation of their spatial structure above 37°C, leading to rapid loss of activity. Irreversible denaturation of their spatial structure occurs above 42°C, greatly limiting their application in high-temperature reaction systems.

[0003] The catalytic activity and ligation efficiency of DNA ligase are not solely determined by the enzyme itself; the buffer system in which it is contained is one of the core factors affecting enzyme function. The core components of a typical DNA ligase buffer include Tris-HCl, MgCl2, and coenzymes (ATP or NAD). + The buffer solution consists of a phosphodiester and a reducing agent. Tris-HCl is used to maintain pH stability. MgCl2 acts as an enzyme activator, regulating the catalytic reaction by binding to the enzyme's active site and DNA substrate. ATP, as a coenzyme, provides energy for phosphodiester bond formation; excessively high concentrations can inhibit blunt-end ligation and even sticky-end ligation, and ATP is easily decomposed, leading to buffer failure due to repeated freeze-thaw cycles. Reducing agents (such as DTT and TCEP) maintain the reduced state of the sulfhydryl groups in the enzyme's active site, stabilizing the enzyme's spatial structure and preventing inactivation due to oxidation. In addition, some buffer solutions contain protein stabilizers such as BSA to reduce enzyme inactivation due to low concentrations and further optimize ligation efficiency.

[0004] Besides the buffer system, factors such as DNA fragment end type, reaction temperature, ligation site characteristics, and reaction system complexity all significantly affect the ligation efficiency and specificity of ligases, forming a multi-factor coupled regulatory network. Regarding end type, sticky ends can spontaneously anneal through base pairing to form a stable double-stranded structure, providing a clear target site for ligase, resulting in significantly higher ligation efficiency than blunt ends. Blunt ends, on the other hand, rely on random collisions of DNA fragments, lacking annealing guidance, and their ligation efficiency is only 1 / 10 to 1 / 100 that of sticky ends, requiring increased enzyme dosage to achieve effective ligation.

[0005] Reaction temperature, as a key regulatory parameter, exhibits a two-way balance in its impact on ligation efficiency: on the one hand, increased temperature enhances the catalytic kinetic efficiency of the enzyme, with the catalytic activity of thermostable ligases reaching its peak at 37°C; on the other hand, high temperatures weaken the base pairing at the sticky ends of DNA, leading to decreased annealing stability and even end-unwinding. Simultaneously, the ligase is prone to irreversible denaturation, which further reduces the actual ligation efficiency. Therefore, conventional sticky-end ligation reactions typically select 16°C as the optimal temperature, balancing enzyme catalytic activity and end-annealing stability; 25°C is considered the upper limit of the effective reaction temperature, retaining over 80% of enzyme activity, but at the cost of decreased end-annealing stability.

[0006] The base composition and pairing state of the ligation site also affect ligation efficiency. Ligases that completely follow Watson-Crick pairing have the highest ligation efficiency, while their ability to ligate mismatched substrates varies depending on the type of mismatch and the type of ligase. For example, T4 DNA ligase has high tolerance for pyrimidine-purine mismatches, while thermostable ligases, although having stronger fidelity, can still ligate mismatched substrates such as T:G and T:T, and have better ability to distinguish upstream base pairs than downstream ones. In complex reaction systems such as multi-fragment assembly and oligonucleotide-assisted ligation, non-specific annealing easily occurs between different DNA fragments and oligonucleotides, forming malligation products that severely interfere with ligation specificity. To solve this problem, the core optimization strategy is to increase the reaction temperature to be close to the melting temperature (Tm) of the DNA fragment. This inhibits the annealing of mismatched fragments through thermal motion while preserving the stability of perfectly paired fragments, thereby improving ligation specificity. However, as mentioned earlier, existing conventional buffer systems cannot support ligases to maintain sufficient activity at higher temperatures. That is, non-thermally stable ligases are rapidly inactivated at high temperatures, while thermostable ligases, although tolerant of high temperatures, suffer from poor substrate adaptability, limited ligation efficiency, and insufficient fidelity. Furthermore, the component ratios of existing buffers cannot be adapted to their high-temperature catalytic requirements.

[0007] NEB's T4 DNA ligase buffer (catalog number B0202S) discloses a composition of 500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, and 100 mM DTT, with a pH of 7.5; however, the optimal operating temperature for this buffer with T4 DNA ligase is 16-25°C. Chinese patent CN116515777A discloses a T4 DNA ligase buffer containing Tris-HCl, MgCl2, and MgCl2. 2+ ATP and DTT, the Mg 2+ The anion is acetate ion; however, the suitable temperature for this bonding system is 25-37℃.

[0008] Therefore, there is an urgent need to develop a buffer system that can provide a stable microenvironment for DNA ligase at higher temperatures and effectively maintain enzyme activity and catalytic efficiency, so as to meet the high-temperature requirements of multi-fragment and highly specific ligation reactions and promote the further application of recombinant DNA technology in complex genome assembly, precise mutation detection and other fields. Summary of the Invention

[0009] This invention addresses the problems existing in the prior art by providing a T4 DNA ligase buffer, the components of which include Tris-HCl, ATP, MgCl2, reduced glutathione, polyvinylpyrrolidone K30, and Triton X-100. This buffer significantly improves the ligation efficiency of T4 DNA ligase at higher temperatures and can be applied to mRNA tailing based on enzyme digestion and ligation.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] On one hand, the present invention provides a buffer for T4 DNA ligase, the components of which include Tris-HCl, ATP, MgCl2, reduced glutathione, polyvinylpyrrolidone K30 and Triton X-100.

[0012] Reduced glutathione is a naturally occurring tripeptide reducing agent with a core structure containing a thiol group (-SH). It can stabilize the spatial structure of enzymes by maintaining the reduced state of the thiol group in the active site of the enzyme, thus preventing the enzyme from becoming inactive due to oxidation.

[0013] Polyvinylpyrrolidone K30 is a linear, non-crosslinked, water-soluble, nonionic polymer with an average molecular weight of approximately 40,000. It is a white powder that readily dissolves in water to form a low-viscosity solution. Its common function is to adsorb trace amounts of polyphenols, polysaccharides, and other impurities in the system through hydrophobic interactions, thereby reducing the non-specific adsorption of nucleic acids and enzymes. Other common molecular weight types of polyvinylpyrrolidone include K25 (average molecular weight of approximately 25,000) and K90 (average molecular weight of approximately 1,300,000).

[0014] Triton X-100 is a nonionic surfactant, chemically known as polyethylene glycol octylphenyl ether. It is a pale yellow, transparent, viscous liquid at room temperature and is readily soluble in water. Its common functions include reducing the surface tension of solutions, promoting the contact between enzymes and DNA substrates, and slightly disrupting the nonspecific aggregation of nucleic acid molecules, thereby improving the homogeneity of the reaction system.

[0015] Preferably, the buffer solution comprises 300-400 mM Tris-HCl, 8-12 mM ATP, 80-120 mM MgCl2, 15-32 mM reduced glutathione, 0.2%-0.4% (w / v) polyvinylpyrrolidone K30 and 0.005%-0.010% (v / v) Triton X-100, with a pH of 7.2-8.0.

[0016] Preferably, the buffer solution comprises 330-380 mM Tris-HCl, 9-11 mM ATP, 90-110 mM MgCl2, 18-30 mM reduced glutathione, 0.25%-0.35% (w / v) polyvinylpyrrolidone K30 and 0.006%-0.009% (v / v) Triton X-100, with a pH of 7.2-7.8.

[0017] In some specific embodiments, the buffer solution comprises 350 mM Tris-HCl, 10 mM ATP, 100 mM MgCl2, 25 mM reduced glutathione, 0.3% (w / v) polyvinylpyrrolidone K30 and 0.008% (v / v) Triton X-100, with a pH of 7.5.

[0018] In some specific embodiments, the buffer solution comprises 300 mM Tris-HCl, 12 mM ATP, 80 mM MgCl2, 32 mM reduced glutathione, 0.2% (w / v) polyvinylpyrrolidone K30 and 0.01% (v / v) Triton X-100, with a pH of 7.2.

[0019] In some specific embodiments, the buffer solution comprises 400 mM Tris-HCl, 8 mM ATP, 120 mM MgCl2, 15 mM reduced glutathione, 0.4% (w / v) polyvinylpyrrolidone K30, 0.005% (v / v) Triton X-100, and pH 8.0.

[0020] On the other hand, the present invention provides a reaction system for T4 DNA ligase, the reaction system comprising the above-mentioned buffer solution, and further comprising a DNA fragment or T4 DNA ligase.

[0021] Preferably, each 10 μl reaction system contains 1 μl of buffer solution.

[0022] In some specific embodiments, the DNA fragments are the enzyme-digested fragment 7-3-2-4 and the enzyme-digested CTNG-PUC57-simple vector from Chinese patent CN120026073A, and these two DNA fragments are linked together by sticky ends.

[0023] The T4 DNA ligases include common T4 DNA ligases and their mutants, and are capable of performing normal T4 DNA ligase activity.

[0024] On the other hand, the present invention provides a kit for DNA ligation, the kit comprising the above-described buffer solution or the above-described reaction system.

[0025] On the other hand, the present invention provides the application of the above-described buffer solution, the above-described reaction system, or the above-described kit in DNA ligation.

[0026] Preferably, the application includes mRNA tailing based on enzyme digestion and ligation.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention provides a T4 DNA ligase buffer, comprising Tris-HCl, ATP, MgCl2, reduced glutathione, polyvinylpyrrolidone K30, and Triton X-100. This buffer significantly improves the ligation efficiency of T4 DNA ligase, and also improves the ligation efficiency at higher temperatures. Reduced glutathione, polyvinylpyrrolidone K30, and Triton X-100 have a synergistic effect and can be used for mRNA tailing based on enzyme digestion and ligation. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the preparation process of fragment 7-3-2-4 in the basic embodiment.

[0030] Figure 2 The plasmid map of the CTNG pUC57-simple vector in the basic embodiment.

[0031] Figure 3 Examples of transformants on different grouped plate culture media corresponding to 30 min of ligation in Example 1 are shown for verification; where A corresponds to the negative control group, B to the positive control group, and C to Example 1. Detailed Implementation

[0032] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0034] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0035] Basic Example: Plasmid Construction and Application

[0036] This invention provides a method for adding a polyA tail to the 3' end of an mRNA molecule, comprising the following steps: using two short single strands containing polyA sequences (containing an "A" of a specific length to be designed), adding BsaI restriction sites to the 3' end of the first sequence and the 5' end of the second sequence, respectively; and adding restriction sites required for subsequent experiments to the 5' end of the first sequence and the 3' end of the second sequence, respectively.

[0037] First, using a primer of about 20bp, the two single strands are separated into two short double strands by T4 DNA Polymerase. Then, the DNA is cut with BsaI enzyme to expose sticky ends with "TTTTT" and "AAAAA".

[0038] The two fragments are then ligated using T4 DNA Ligase to obtain the ligated product, which is a long double strand containing the required amount of polyA and restriction enzyme sites for the experiment.

[0039] Double enzyme digestion allows the polyA fragment containing a specific number of bases to be inserted into the desired vector, and after transcription, 3' tailed mRNA containing a specific number of bases is obtained.

[0040] The specific methods are as follows: Unless otherwise specified, the equipment and materials used in the basic examples can be easily obtained from commercial companies. Specifically, some of the experimental reagents and equipment involved include: synthetic single-stranded oligo (Sangon Biotech (Shanghai) Co., Ltd.), T4 DNA Polymerase (NEB), T4 DNA Ligase (NEB), BsaI restriction endonuclease: BsaI-HF (NEB), EcoRI restriction endonuclease: EcoRI-HF (NEB), HindIII restriction endonuclease: HindIII-HF (NEB), MiniBest gel recovery kit (TaKaRa), CTNG pUC57-simple vector (synthesized by GenScript Biotech Co., Ltd.), competent cells (Takara Stable Competent Cells, purchased from TaKaRa), universal DNA recovery kit (Tiangen Biotech (Beijing) Co., Ltd.); PCR amplification instrument, ultra-high speed centrifuge, clean bench, constant temperature shaking incubator, 37℃ biochemical incubator, electrophoresis apparatus, and gel imaging system.

[0041] For clarity, refer to steps 1-4. Figure 1 The preparation process flow chart.

[0042] Step 1: Using two short single strands containing polyA sequences, BsaI restriction sites were added to the 3' end of the first sequence and the 5' end of the second sequence, respectively; restriction sites (HindIII restriction site and EcoRI restriction site) were added to the 5' end of the first sequence and the 3' end of the second sequence, respectively, to obtain OLG-7 and OLG-2, respectively. The primer names and sequences are shown in Table 1. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] Table 1 Primer Sequence Description

[0044]

[0045] It should be noted that in Table 1,

[0046] The OLG-7 with a complete double strand: after digestion with BsaI, a 60nt "A" is exposed, with a HindIII restriction site at 5' and a BsaI restriction site at 3'.

[0047] The OLG-2 with complete double strands: after digestion with BsaI, a 60nt "A" is exposed, with a BsaI cleavage site at 5' and an EcoRI cleavage site at 3'.

[0048] OLG-3: Used to complete double chains in OLG-7.

[0049] OLG-4: Used to complete the double chain of OLG-2.

[0050] Step 2:

[0051] Reference Figure 1 Using primers (OLG-3 and OLG-4), two short single strands (OLG-7 and OLG-2) were converted into two short double strands under the action of T4 DNA Polymerase. The short double strands were identified to confirm that double strands had been formed. Then, the DNA was cut with BsaI enzyme to expose sticky ends with "TTTTT" and "AAAAA".

[0052] Specifically, the reaction system for the formation of short single strands into short double strands is shown in Tables 2 and 3. The reaction conditions were: incubate the reactants in a PCR amplification instrument at 12°C for 15 min. The reaction was terminated by incubating in a metal bath at 75°C for 20 min.

[0053] Table 2 Reaction system of OLG-7 and OLG-3

[0054]

[0055] Table 3 Reaction system of OLG-2 and OLG-4

[0056]

[0057] After the reaction, short double strands were formed and identified: 300 ng each of the two short double strands, OLG-7, and OLG-2 were subjected to polyacrylamide gel electrophoresis. The electrophoresis results showed that the two short single strands (OLG-7 and OLG-2) did not show bands; the short double strand formed by OLG-7 and OLG-3 had extraneous bands; and the short double strand formed by OLG-2 and OLG-4 produced a single band. Therefore, both OLG-7 and OLG-2 formed short double strands, which were named 7-3 and 2-4, respectively.

[0058] The formed short double strands (7-3 and 2-4) were digested with BsaI-HF. The digestion system is shown in Tables 4 and 5. The reaction conditions were 37℃ for 1 h.

[0059] Table 4 Enzyme digestion system for short double-stranded (7-3)

[0060]

[0061] Table 5. Enzyme digestion system for short double-stranded (2-4) strands

[0062]

[0063] Step 3: Ligate the short double strands obtained in Step 2 with T4 DNA Ligase. The ligation product is a long double strand, which contains the required number of polyA molecules and restriction enzyme sites as designed in the experiment.

[0064] Specifically, the short double strands (7-3 and 2-4) were ligated using T4 DNA Ligase. Since 10×rCutSmart Buffer was used in the previous step, ATP (as required by the enzyme reaction) needed to be added to the ligation reaction system. The ligation reaction system is shown in Table 6. Reaction conditions: PCR incubation at 25℃ for 2 hours, followed by heat inactivation at 65℃ for 10 minutes to prevent affecting the efficiency of the next enzyme digestion step. The long double strand was named 7-3-2-4 or fragment 7-3-2-4, containing 120 bp of polyA. The resulting polyA fragment has EcoRI and HindIII restriction sites at both ends.

[0065] Table 6 Connection Reaction System

[0066]

[0067] The sequence of 7-3-2-4 (SEQ ID No. 5) is as follows:

[0068] 5'-CGGGAAAGCTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATTCGGATC-3'.

[0069] Step 4: Ligate the digested fragment 7-3-2-4 and the digested CTNG-PUC57-simple vector using T4 DNA Ligase to obtain the backbone. Double digestion of the backbone yields a polyA fragment containing a specific number of bases. Inserting this polyA fragment into the desired vector and transcribing it yields an mRNA with a 3' tail containing the specific number of bases. The specific method is as follows:

[0070] (1) The vector for inserting the fragment is the CTNG pUC57-simple vector. Information about the CTNG pUC57-simple vector: The target sequence CTNG was synthesized, and pUC57-simple was inserted through the EcoRV site to obtain the CTNG pUC57-simple vector. The vector map is shown below. Figure 2 The target sequence was designed by adding T7 Promoter and SP6 Promoter before it (a conventional method). The CTNG pUC57-simple vector was synthesized by Qingke Biotechnology. It should be noted that the target sequence CTNG does not affect the technical effect of the technical solution of this application and is not a key influencing factor, therefore its sequence is not specifically specified.

[0071] (2) The fragment 7-3-2-4 and the CTNG pUC57-simple vector were double-digested with EcoRI restriction endonuclease and HindIII restriction endonuclease.

[0072] The enzyme digestion system for fragment 7-3-2-4 is shown in Table 7. Reaction conditions: 1 hour at 37°C.

[0073] Table 7 Enzyme digestion system for fragment 7-3-2-4

[0074]

[0075] The enzyme digestion products were recovered using the MiniBest gel extraction kit (TaKaRa) to remove inorganic salts and small fragments. Fragment recovery was performed by centrifugation at room temperature, following the instructions of the aforementioned kit: 132 μl of sol buffer was added, followed by 45 μl of isopropanol, and mixed thoroughly. The recovered product was then identified as a single band by agarose gel electrophoresis.

[0076] The enzyme digestion system of the CTNG pUC57-simple vector is shown in Table 8. Reaction conditions: 1 h at 37°C.

[0077] Table 8 Enzyme digestion system of CTNG pUC57-simple vector

[0078]

[0079] The CTNG pUC57-simple vector was digested with enzymes and then excised using a gel. The digestion products were recovered using a universal DNA recovery kit to remove inorganic salts and small fragments. Fragment recovery was performed by centrifugation at room temperature, following the experimental procedure described in the aforementioned kit. The recovered product was then identified as a single band by agarose gel electrophoresis.

[0080] The digested fragment 7-3-2-4 and the digested CTNG-PUC57-simple vector were ligated using T4 DNA Ligase to obtain the backbone. The ligation system is shown in Table 9. Ligation conditions: ligation at 25℃ for more than 1 hour (incubation in a PCR instrument).

[0081] Table 9 Connection System

[0082]

[0083] The T4 DNA ligase was purchased from NEB (catalog number M0202L); the T4 DNA ligase buffer was purchased from NEB (catalog number B0202S), and its components were 500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP and 100 mM DTT, pH 7.5.

[0084] Competent cells were transformed using a scaffold, and clones were selected for sequencing to verify sequence accuracy.

[0085] The backbone prepared in step 4 is a backbone containing a specific number of polyA bases and restriction enzyme sites, namely a CTNG pUC57-simple vector containing continuous polyA bases. The backbone is double-digested to obtain a polyA fragment containing a specific number of bases. The polyA fragment containing a specific number of bases is inserted into a plasmid containing a specific gene, and after linearization, it is transcribed to obtain a 3' tailed mRNA containing a specific number of bases (for specific methods, please refer to CN120026073A).

[0086] Example 1: T4 DNA ligase buffer

[0087] This embodiment provides a T4 DNA ligase buffer with the following components: 350mM Tris-HCl, 10mM ATP, 100mM MgCl2, 25mM reduced glutathione, 0.3% (w / v) polyvinylpyrrolidone K30, and 0.008% (v / v) Triton X-100, with a pH of 7.5.

[0088] Example 2: T4 DNA ligase buffer

[0089] This embodiment provides a T4 DNA ligase buffer with the following components: 300mM Tris-HCl, 12mM ATP, 80mM MgCl2, 32mM reduced glutathione, 0.2% (w / v) polyvinylpyrrolidone K30, 0.01% (v / v) Triton X-100, and pH 7.2.

[0090] Example 3: T4 DNA ligase buffer

[0091] This embodiment provides a T4 DNA ligase buffer with the following components: 400mM Tris-HCl, 8mM ATP, 120mM MgCl2, 15mM reduced glutathione, 0.4% (w / v) polyvinylpyrrolidone K30, 0.005% (v / v) Triton X-100, and pH 8.0.

[0092] Comparative Example 1: T4 DNA Ligase Buffer

[0093] A T4 DNA ligase buffer, differing from Example 1 only in that it does not contain polyvinylpyrrolidone K30 and Triton X-100, otherwise it is the same as Example 1.

[0094] Comparative Example 2: T4 DNA Ligase Buffer

[0095] A T4 DNA ligase buffer, differing from Example 1 only in that reduced glutathione and Triton X-100 are not added; otherwise, it is the same as Example 1.

[0096] Comparative Example 3: T4 DNA Ligase Buffer

[0097] A T4 DNA ligase buffer, differing from Example 1 only in that it does not contain reduced glutathione and polyvinylpyrrolidone K30, otherwise it is the same as Example 1.

[0098] Comparative Example 4: T4 DNA Ligase Buffer

[0099] A T4 DNA ligase buffer, differing from Example 1 only in that reduced glutathione, polyvinylpyrrolidone K30, and Triton X-100 are not added; otherwise, it is the same as Example 1.

[0100] Comparative Example 5: T4 DNA Ligase Buffer. A T4 DNA ligase buffer, which differs from Example 1 only in that reduced glutathione is replaced with an equimolar amount of DTT, otherwise it is the same as Example 1.

[0101] Comparative Example 6: T4 DNA Ligase Buffer. A T4 DNA ligase buffer, differing from Example 1 only in that polyvinylpyrrolidone K30 is replaced with an equal mass of polyvinylpyrrolidone K25, otherwise the same as Example 1.

[0102] Comparative Example 7: T4 DNA Ligase Buffer

[0103] A T4 DNA ligase buffer is provided, which differs from Example 1 only in that Triton X-100 is replaced with an equal volume of Tween-80, otherwise it is the same as Example 1.

[0104] Comparative Example 8: T4 DNA Ligase Buffer

[0105] A T4 DNA ligase buffer is provided, which differs from Example 1 only in that the reduced glutathione concentration is 45 mM, and all other aspects are the same as in Example 1.

[0106] Comparative Example 9: T4 DNA Ligase Buffer

[0107] A T4 DNA ligase buffer, differing from Example 1 only in that the concentration of polyvinylpyrrolidone K30 is 0.6% (w / v), otherwise the same as Example 1.

[0108] Comparative Example 10: T4 DNA Ligase Buffer

[0109] A T4 DNA ligase buffer, differing from Example 1 only in that the Triton X-100 concentration is 0.015% (v / v), otherwise identical to Example 1.

[0110] Example 1: Application of T4 DNA ligase buffer

[0111] Following the method of the basic embodiment, the ligase buffer in the ligation system of Table 9 was replaced with the ligase buffer of the example or comparative example, while other components remained unchanged. The fragment 7-3-2-4 digestion recovery product and the digested CTNG-PUC57-simple vector were ligated respectively. The ligase buffer (specifically composed of: 500mM Tris-HAc, 100mM Mg(OAc)2, 10mM ATP, 100mM DTT, pH 7.5; i.e. Buffer3 in Example 1 of Chinese Patent CN116515777A) was used as a positive control for the ligase buffer, and the reaction system without the addition of the ligase buffer (using nuclease-free water to replace the ligase buffer) was used as a negative control for the ligase buffer.

[0112] (1) Connection efficiency test

[0113] Different ligation times (n=10) were set for each reaction system. Specifically, the ligation reaction temperature was 25℃, and the reaction termination solution (EDTA, final concentration 10mM) was added at ligation times of 5 min, 30 min, and 90 min. The system was stored at 4℃, and the ligation efficiency of different ligase buffers was determined by the conversion efficiency method.

[0114] Ligation efficiency can be quantified by colony-forming units (CFU) after transformation of competent cells. The principle is that the higher the ligation efficiency, the more positive recombinant vectors there are, and the more colonies there are after transformation.

[0115] Operating steps:

[0116] Take equal volumes of ligation reaction solution (5 μl) and transform them into competent cells of the same batch and titer; specifically, use DH5α competent cells (titer 1×10⁻⁶). 8 The ligation product (CFU / μg) was serially diluted 10-fold, and 100 μl was spread onto LB medium containing ampicillin and incubated at 37°C for 12 h. Figure 3 Examples of transformants on plate culture media corresponding to different groups (negative control group, positive control group, and Example 1) after 30 minutes of ligation are shown in Table 10. (The images were taken with ordinary equipment, which is sufficient to distinguish the obvious differences in the number of transformants between different groups.) The CFU values ​​of transformants corresponding to different buffer solutions and different ligation times were statistically analyzed. Compared with Comparative Example 4, Comparative Examples 1-3 improved the ligation efficiency. Example 1 further significantly improved the ligation efficiency based on Comparative Examples 1-3. The calculation of the improvement showed that reduced glutathione, polyvinylpyrrolidone K30, and Triton X-100 in the buffer solution had a synergistic effect.

[0117] Table 10. CFU values ​​of transformants at different ligation times in different buffer solutions (n=10)

[0118]

[0119] Note: In Table 10, data in the same column are marked with the same letter to indicate no significant difference (P>0.05), and data marked with different letters to indicate significant difference (P<0.05).

[0120] (2) Connection temperature test

[0121] Different ligation temperatures (n=10) were set for each reaction system. Specifically, three temperatures were set: 25℃, 37℃, and 45℃. After ligation reaction for 30 min, reaction termination solution (EDTA, final concentration 10 mM) was added, and the system was stored at 4℃. The ligation efficiency of different ligase buffers was determined by the conversion efficiency method.

[0122] Operating steps:

[0123] Take an equal volume of ligation reaction solution (5 μl) and transform it into competent cells of the same batch and titer; specifically, use DH5α competent cells (titer 1×10⁻⁶). 8The ligation products were serially diluted 10-fold, and 100 μl of each product was spread on LB medium containing ampicillin and incubated at 37°C for 12 h. The CFU values ​​of the transformants corresponding to different buffers and ligation temperatures were counted, and the results are shown in Table 11. Compared with the comparative and positive controls, Examples 1-3 maintained high ligation efficiency at higher temperatures (37°C and 45°C). Specifically, based on the data at 25°C, the ligation efficiency of Examples 1-3 was approximately 80% at 37°C and approximately 75% at 45°C, while the ligation efficiency of the comparative and positive controls was approximately 65%-80% at 37°C and approximately 50%-70% at 45°C.

[0124] Table 11. CFU values ​​of transformants at different buffer solutions and ligation temperatures (n=10)

[0125]

[0126] Note: In Table 11, data in the same column are marked with the same letter to indicate no significant difference (P>0.05), and data marked with different letters to indicate significant difference (P<0.05).

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A buffer for T4 DNA ligase, characterized in that, The composition consists of 300-400mM Tris-HCl, 8-12mM ATP, 80-120mM MgCl2, 15-32mM reduced glutathione, 0.2%-0.4% (w / v) polyvinylpyrrolidone K30 and 0.005%-0.010% (v / v) Triton X-100, with a pH of 7.2-8.

0.

2. The buffer solution according to claim 1, characterized in that, The composition consists of 330-380 mM Tris-HCl, 9-11 mM ATP, 90-110 mM MgCl2, 18-30 mM reduced glutathione, 0.25%-0.35% (w / v) polyvinylpyrrolidone K30 and 0.006%-0.009% (v / v) Triton X-100, with a pH of 7.2-7.

8.

3. The buffer solution according to claim 2, characterized in that, The composition consists of 350 mM Tris-HCl, 10 mM ATP, 100 mM MgCl2, 25 mM reduced glutathione, 0.3% (w / v) polyvinylpyrrolidone K30 and 0.008% (v / v) Triton X-100, with a pH of 7.

5.

4. The buffer solution according to claim 1, characterized in that, The composition consists of 300 mM Tris-HCl, 12 mM ATP, 80 mM MgCl2, 32 mM reduced glutathione, 0.2% (w / v) polyvinylpyrrolidone K30 and 0.01% (v / v) Triton X-100, with a pH of 7.

2.

5. The buffer solution according to claim 1, characterized in that, The composition consists of 400 mM Tris-HCl, 8 mM ATP, 120 mM MgCl2, 15 mM reduced glutathione, 0.4% (w / v) polyvinylpyrrolidone K30, and 0.005% (v / v) Triton X-100, with a pH of 8.

0.

6. A reaction system for T4 DNA ligase, characterized in that, The reaction system comprises the buffer solution according to any one of claims 1-5, and further comprises a DNA fragment or T4 DNA ligase.

7. The reaction system according to claim 6, characterized in that, Each 10 μl reaction system contains 1 μl of buffer.

8. A kit for DNA ligation, characterized in that, The kit comprises the buffer solution according to any one of claims 1-5 or the reaction system according to any one of claims 6-7.

9. The use of the buffer solution according to any one of claims 1-5, the reaction system according to any one of claims 6-7, or the kit according to claim 8 in DNA ligation, characterized in that, The applications include mRNA tailing based on enzyme digestion and ligation.

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