Buffer solution, kit and reaction system for nucleic acid cleavage and application

Abstract

The invention relates to a buffer solution, a kit and a reaction system for nucleic acid cleavage and application. The buffer solution is prepared from the following components with working concentration: 10mM to 50mM of Tris-HCl, 10mM to 100mM of KCl and 1mM to 50mM of MgCl2. Comprising the buffer solution. By using the buffer solution provided by the invention, oligonucleotide can be cut from a solid phase at a predetermined site, and the cutting effect is good.

Description

Technical Field

[0001] The present application relates to the field of biochemical technology, and specifically relates to a buffer, a kit, a reaction system for nucleic acid cleavage, and applications thereof. Background Art

[0002] DNA synthesis technologies mainly include chemical methods and biological methods. Among them, the chemical method (especially the solid-phase phosphoramidite triester synthesis method) is the most mature and widely used, while the biological method has emerged abroad but is still in the principle verification stage. The DNA synthesis technology by bioenzymes is usually carried out in an aqueous environment, which can effectively avoid problems such as the continuously increasing error rate during the synthesis process of the chemical synthesis method as the chain extends, and is expected to synthesize longer DNA molecules at a lower cost.

[0003] Bioenzymatic methods can synthesize DNA in a solid-phase synthesis manner. Solid-phase synthesis usually uses magnetic beads labeled with streptavidin as a carrier, and uses oligonucleotides labeled with biotin to bind to it. After completing one round of reaction, the oligonucleotides on the solid phase can be effectively separated from other reactants. After the last round of synthesis reaction is completed, the oligonucleotides need to be cleaved from the solid phase. The traditional method uses toxic chemical reagents to cleave the oligonucleotides from the solid phase. To avoid the use of toxic chemical reagents, a reaction system and method that are safe, non-toxic, and have good cleavage effects are needed. Summary of the Invention

[0004] Based on this, an embodiment of the present application provides a buffer, a kit, and a reaction system for nucleic acid cleavage. Using the buffer, kit, and reaction system of the present application can cleave oligonucleotides from the solid phase with good cleavage effects.

[0005] The technical solutions are as follows:

[0006] A buffer for nucleic acid cleavage, the buffer is composed of components at the following working concentrations: 10 mM - 50 mM Tris-HCl, 10 mM - 100 mM KCl, 1 mM - 50 mM MgCl2, and water.

[0007] A kit for nucleic acid cleavage, the kit includes the buffer, UDG enzyme, and APE enzyme.

[0008] A reaction system for nucleic acid cleavage, the reaction system includes the buffer, the UDG enzyme and APE enzyme in the kit, nucleic acid, and water.

[0009] In one embodiment, the nucleic acid includes ssDNA or solid-phase linked DNA; uracil bases are incorporated at the pre-positioning sites of the ssDNA or solid-phase linked DNA as cleavage sites.

[0010] In one embodiment, every 50 μL of the reaction system includes 5 μL of 10× the buffer, 1 μL - 20 μL of the stock solution of UDG enzyme diluted 20-fold, 0.5 μL - 8 μL of APE enzyme at 0.4 μg / μL - 1 μg / μL, 100 pmol - 400 pmol of nucleic acid, and water.

[0011] Compared with the traditional technology, the present application has the following beneficial effects:

[0012] The buffer provided by the present application can improve the activity and enzymatic cleavage efficiency of UDG enzyme and / or APE enzyme; based on this buffer, UDG enzyme and APE enzyme, a kit and a reaction system can be provided for nucleic acid cleavage or cutting nucleic acid from a solid phase at a predetermined cleavage site, with good cutting effect, simple operation, and being safe and non-toxic. Description of the Drawings

[0013] Figure 1 SDS-PAGE detection diagram for U-1 to U-6 enzymes; Note: line1: original bacteria; line2: induction; line3: supernatant; line4: precipitate; line5: empty Ni; line6: 50 mM eluted Ni; line7: 300 mM eluted sample; line8: 500 mM eluted Ni;

[0014] Figure 2 SDS-PAGE detection diagram for A-1 to A-5 enzymes; Note: line1: original bacteria; line2: induction; line3: supernatant; line4: precipitate; line5: empty Ni; line6: 50 mM eluted Ni; line7: 300 mM eluted sample; line8: 500 mM eluted Ni;

[0015] Figure 3 Detection diagram for NaOH treatment temperature and time;

[0016] Figure 4 Detection diagram for UDG types and reaction concentrations;

[0017] Figure 5 Detection diagram for UDG types, reaction concentrations, and reaction times;

[0018] Figure 6 Detection diagram for U-3 reaction concentrations and reaction times;

[0019] Figure 7 Detection diagram for reaction buffer optimization; where, in the upper figure, U represents UDG Buffer, A represents APE Buffer, and B represents the improved buffer; in the lower figure, in the one-step method and two-step method, U + B represents the mixture of UDG Buffer and the improved buffer;

[0020] Figure 8 Detection chart for APE species and reaction concentration;

[0021] Figure 9 Detection chart for DTT optimization;

[0022] Figure 10 Detection chart for MgCl2 concentration optimization;

[0023] Figure 11 Detection chart for KCl, Tris-HCl, PCNA, and BSA concentration optimization;

[0024] Figure 12 Detection chart for one-step reaction temperature optimization;

[0025] Figure 13 Detection chart for one-step reaction time optimization. Specific implementation manners

[0026] To make the above objects, features, and advantages of the present application more obvious and understandable, the specific implementation manners of the present application are described in detail below. Many specific details are set forth in the following description in order to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0028] To avoid using traditional methods to cleave the DNA after solid-phase synthesis from the solid phase, the present application uses a double-enzyme method for cleavage, including uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease (APE). Among them, uracil-DNA glycosylase is responsible for removing uracil from DNA by hydrolyzing the N-glycosidic bond that links the base to the deoxyribose backbone, leaving an apyrimidinic (AP) site to be cleaved by apurinic / apyrimidinic endonuclease at the 3' or 5' end of the AP site, thereby releasing the oligonucleotide. Therefore, when synthesizing DNA on a solid phase, it is necessary to incorporate uracil deoxynucleotide at the pre-determined site as the cleavage site to cleave the oligonucleotide from the solid phase.

[0029] This application expresses and screens uracil-DNA glycosylase and apurinic / apyrimidinic endonuclease, and determines the cleavage efficiency and parameters by optimizing the reaction system, reaction conditions, termination conditions, etc., for cleaving the solid-phase synthesized DNA from the solid phase.

[0030] Based on this, an embodiment of this application provides a buffer, which includes components at the following working concentrations: 10 mM to 50 mM Tris-HCl, 10 mM to 100 mM KCl, and 1 mM to 50 mM MgCl2. This buffer improves the activity and cleavage efficiency of UDG enzyme and / or APE enzyme.

[0031] In a specific example, the buffer includes 10 mM to 30 mM Tris-HCl, 10 mM to 50 mM KCl, and 5 mM to 20 mM MgCl2, wherein the pH value of Tris-HCl is 7.8 to 8.8.

[0032] In a preferred specific example, the buffer includes 20 mM Tris-HCl, 20 mM KCl, and 10 mM MgCl2, wherein the pH value of Tris-HCl is 8.1.

[0033] An embodiment of this application also provides a kit for nucleic acid cleavage, which includes the above buffer, UDG enzyme, and APE enzyme.

[0034] This application expresses uracil-DNA glycosylase (abbreviated as UDG enzyme or UNG enzyme) and apurinic / apyrimidinic endonuclease, and screens out enzymes with high ssDNA cleavage activity.

[0035] In a specific example, the UDG enzyme includes the UDG enzyme with the sequence shown in at least one of SEQ ID NO.1 to SEQ ID NO.6.

[0036] In a specific example, the UDG enzyme includes the UDG enzyme with the sequence shown in at least one of SEQ ID NO.1 to 2 and SEQ ID NO.6.

[0037] In a preferred specific example, the UDG enzyme includes the UDG enzyme with the sequence shown in at least one of SEQ ID NO.3 to SEQ ID NO.5. In a more preferred specific example, the UDG enzyme includes the UDG enzyme with the sequence shown in SEQ ID NO.3 and / or SEQ ID NO.5.

[0038] In a specific example, the APE enzyme includes the APE enzyme with the sequence shown in at least one of SEQ ID NO.7 to SEQ ID NO.11.

[0039] In a preferred specific example, the APE enzyme includes the APE enzyme having a sequence shown in at least one of SEQ ID NO.7 and SEQ ID NOs.9 to 11. In a more preferred specific example, the APE enzyme includes the APE enzyme having the sequence shown in SEQ ID NO.9.

[0040] One embodiment of the present application provides a reaction system for nucleic acid cleavage, which reaction system includes the above-mentioned buffer, the UDG enzyme and the APE enzyme in the above-mentioned kit, nucleic acid and water. Wherein, the nucleic acid includes ssDNA or solid-phase linked DNA; uracil deoxynucleotide is incorporated at a pre-determined site of the ssDNA or solid-phase linked DNA as a cleavage site. At least one uracil deoxynucleotide is incorporated, and those skilled in the art can incorporate it at a specific site according to the cleavage requirement. Incorporating uracil deoxynucleotide during DNA synthesis can adopt conventional technical means in the art.

[0041] Exemplary ssDNA is 5’-GTATTGCCTTTCCTACGACCTCACGC(dU)TAAT

[0042] GAATGACACTCAATGCACAT-3’, SEQ ID NO.12.

[0043] In a specific example, every 50 μL of the above-mentioned reaction system includes 5 μL of 10× the above-mentioned buffer, 0.5 μL to 8 μL of APE enzyme at 0.4 μg / μL to 1 μg / μL, 1 μL to 20 μL of 20-fold diluted UDG enzyme stock solution, 100 pmol to 400 pmol of nucleic acid and water.

[0044] In a specific example, every 50 μL of the above-mentioned reaction system includes 5 μL of 10× the above-mentioned buffer, 1 μL to 7 μL of APE enzyme at 0.5 μg / μL, 1 μL to 5 μL of 20-fold diluted UDG enzyme stock solution, 200 pmol to 400 pmol of nucleic acid and water.

[0045] In a preferred specific example, every 50 μL of the above-mentioned reaction system includes 5 μL of 10× the above-mentioned buffer, 2 μL of APE enzyme at 0.5 μg / μL, 1 μL of 20-fold diluted UDG enzyme stock solution, 200 pmol of nucleic acid and water.

[0046] One embodiment of the present application provides the use of the above-mentioned buffer or the above-mentioned kit or the above-mentioned reaction system in the preparation of a product for cleaving nucleic acid from a solid phase.

[0047] One embodiment of the present application provides a method for cleaving ssDNA or cleaving nucleotides from a solid phase, which method comprises cleaving using the above-mentioned buffer solution, the above-mentioned kit or the above-mentioned reaction system. Using the buffer solution, kit and reaction system provided by the present application to cleave nucleic acids from a solid phase has a higher effect and is more complete in cleavage within a short time.

[0048] In a specific example, the cleavage conditions include reacting at 37°C to 55°C for 2 min to 30 min, and then terminating the reaction with EDTA.

[0049] In a preferred specific example, the cleavage conditions include reacting at 37°C for 5 min, and then terminating the reaction with EDTA. EDTA can bind metal ions to inactivate the enzyme and play a terminating role. The working concentration of EDTA is 8 mM to 12 mM, optionally 10 mM.

[0050] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following examples, preference is given to the guidance given in the present application, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0051] In the following specific examples, for the measurement parameters of the raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0052] Example 1

[0053] I. Design and synthesis of UDG and APE enzymes (1) The main materials involved in this example are as follows:

[0054] 1. Experimental instruments

[0055] PCR instrument, laminar flow hood, shaker, ultrasonic crusher, centrifuge, refrigerated centrifuge, protein purifier, chromatography cabinet, electrophoresis instrument, electrophoresis tank, gel imager and microwave oven.

[0056] 2. Experimental consumables

[0057] Pipette, disposable tips, EP tubes, disposable plates, power strips, test tubes, shake flasks, beakers, centrifuge tubes, centrifuge cups, protein purification magnetic beads, protein purification columns, nickel fillers.

[0058] 3. Preparation of experimental reagents

[0059] 3.1 Fermentation medium, as shown in Table 1:

[0060] Table 1

[0061]

[0062] 3.2 Protein purification buffer

[0063] 3.2.1 Equilibration buffer, as shown in Table 2:

[0064] Table 2

[0065]

[0066] 3.2.2 Washing buffer, as shown in Table 3:

[0067] Table 3

[0068]

[0069] 3.2.3 Elution buffer, as shown in Table 4:

[0070] Table 4

[0071]

[0072] 3.3 Protein dialysis buffer, as shown in Table 5:

[0073] Table 5

[0074]

[0075]

[0076] 4. Experimental strains

[0077] E.coli top10, E.coli BL21(DE3).

[0078] 5. Experimental vectors

[0079] pET series

[0080] (II) Experimental methods

[0081] 1. Gene design and synthesis

[0082] In order to test the soluble expression levels and enzyme activities of UDG and APE from different species sources, sequences from different sources or different mutants were selected for design and synthesis. After screening for highly active UDG and APE, the reaction systems and conditions were optimized to obtain the optimal reaction conditions, laying a foundation for the screening of DNA cleavage research in solid-phase synthesis in the later stage.

[0083] After the gene sequences were designed, gene synthesis (synthesized by Nanjing Tsingke Biotechnology Co., Ltd.) and codon optimization were carried out and ligated to plasmids to obtain recombinant expression vectors for UDG and APE, as shown in Table 6.

[0084] Table 6

[0085] Serial Number Gene Name Plasmid Resistance Nucleotide Sequence U-1 UNG(E) pET-28a Kan+ SEQ ID NO.1 U-2 UNG(DR) pET-28a Kan+ SEQ ID NO.2 U-3 UNG(H R276) pET-28a Kan+ SEQ ID NO.3 U-4 UNG(H R276C) pET-28a Kan+ SEQ ID NO.4 U-5 UNG(H R276H) pET-28a Kan+ SEQ ID NO.5 U-6 UNG(HSVP) pET-28a Kan+ SEQ ID NO.6 A-1 Tko EndoQ pET-28a Kan+ SEQ ID NO.7 A-2 (9°N)EndoQ pET-28a Kan+ SEQ ID NO.8 A-3 Endonuclease IV(E) pET-28a Kan+ SEQ ID NO.9 A-4 Endonuclease IV(TE) pET-28a Kan+ SEQ ID NO.10 A-5 Endonuclease IV(Pf) pET-28a Kan+ SEQ ID NO.11

[0086] 2. Induced expression

[0087] Extract the UDG or APE recombinant expression plasmid from E. coli top10, and slowly add 5 - 10 μL respectively to the competent cells of E. coli BL21(DE3). After mixing, incubate on ice for 30 min, heat shock at 42 °C for 45 - 90 s, and then incubate on ice for 1 - 2 min. Add 900 μL of LB and incubate at 37 °C with shaking at 220 rpm for 1 h. Centrifuge at 5000 rpm for 3 min, leave a little supernatant, pipette and mix well, then spread on the LB plate medium containing kanamycin resistance, and incubate inverted at 37 °C overnight. Pick the above monoclonal colonies and inoculate them into 5 mL of LB medium containing kanamycin resistance, culture overnight at 37 °C with shaking at 220 rpm. Inoculate the bacterial solution into 100 mL of LB medium containing kanamycin resistance at an inoculation amount of 1%, culture at 37 °C with shaking at 220 rpm until the OD reaches 0.6 - 0.8, add IPTG with a final concentration of 0.5 mM, and induce overnight at 16 °C with shaking at 120 rpm.

[0088] 3. Protein purification

[0089] 3.1 Cell disruption

[0090] After the induced expression is completed, take 500 mL of the bacterial solution, centrifuge at 3500 rpm for 15 min, discard the supernatant, resuspend with 40 mL of equilibration buffer, add a protease inhibitor with a final concentration of 1 mM, use an ultrasonic cell disruptor to lyse the cells, the disruption temperature is 4 °C, the power is 40%, the ultrasonic time is 3 s, the intermittent time is 5 s, and the total duration is 60 min. After disruption, centrifuge at 8000 rpm for 60 min at 4 °C, and collect the supernatant.

[0091] 3.2 Nickel column equilibration and purification

[0092] First, rinse the nickel column with 10 ml of sterile water, and then equilibrate the nickel column with 10 ml of equilibration buffer.

[0093] Load the sample onto the column and let it pass through twice. Then elute the impurity proteins with the washing buffer containing 50 mM imidazole. Incubate for 15 min for the first time and then elute. The total elution volume is 20 ml for two times. Take 10 μL of the sample and run SDS-PAGE for standby.

[0094] Then incubate with the washing buffer containing 300 mM imidazole for 15 min and then elute. Each elution volume is 4 ml, and a total of 8 mL of the target protein is collected and collected in a new 15 mL tube. Take 40 μL of the sample and run SDS-PAGE for standby.

[0095] Finally, use the elution buffer containing 500 mM imidazole to remove all proteins on the column until the Coomassie detection solution no longer changes color. Then collect the sample BAE, and rinse the nickel column with 20 ml of sterile water and then with 10 ml of 20% ethanol.

[0096] 4. SDS-PAGE Detection

[0097] Use a 4%-20% SDS-PAGE precast gel (provided by Hubei Qingke Biotechnology Co., Ltd.) and place it in the electrophoresis tank for gel running. Take different purified samples, add Loading buffer and mix well. Load 20 μL of the sample and 5 μL of the Marker. Run the gel at 160 V for 30 min. Stain the gel by heating with the staining solution for 15 min. After cooling, use a gel imager to take pictures. Analyze whether the size of the target protein band is correct with reference to the size of the Marker band.

[0098] 5. Protein Concentration Determination

[0099] After washing with ultrapure water, zero with the elution buffer, and then use SAM 4000 to measure the concentration of the purified target protein and the 260 / 280 value.

[0100] 6. Protein Dialysis and Preservation

[0101] Load the protein into a dialysis bag with a molecular weight cut-off of 10 kDa, and clamp both ends of the dialysis bag with a sealing clip. Place it in the pre-prepared dialysis buffer and put it on the magnetic stirrer in the chromatography cabinet for overnight dialysis.

[0102] Take out the dialyzed protein the next morning, load it into a centrifuge tube, and freeze it at -80 °C for later use.

[0103] (III) Experimental Results

[0104] The SDS-PAGE detection results of the purified UDG and APE enzymes are as Figure 1 and Figure 2 shown, and the target protein can be obtained after purification.

[0105] II. Optimization of the Reaction Conditions for ssDNA Cleavage (I) Screening of UDG Enzyme and Optimization of Its Dosage

[0106] After the UDG enzyme converts dU on ssDNA into an apurinic and apyrimidinic site, it is prone to break under high-temperature NaOH conditions. Since the broken fragments carry a fluorescent group, the catalytic activity of UDG can be detected by SDS-PAGE. Therefore, the termination temperature and time of NaOH are optimized first and then used for the screening of UDG enzyme.

[0107] 1. Optimization of the Termination Temperature and Time of NaOH

[0108] 1.1 Optimization of NaOH termination temperature

[0109] Prepare the reaction system, and the specific formula is shown in Table 7, where 10×buffer is shown in Table 12.

[0110] Table 7

[0111]

[0112] Reaction conditions: React at 37°C for 30 min, and treat with 50 mM NaOH at the final concentration under high-temperature conditions of 45°C, 55°C, 65°C, 75°C, 85°C, and 95°C for 5 min.

[0113] 1.2 Optimization of NaOH termination time

[0114] Prepare the reaction system, and the specific formula is shown in Table 8, where 10×buffer is shown in Table 12.

[0115] Table 8

[0116]

[0117] Reaction conditions: React at 37°C for 30 min, and treat with 50 mM NaOH at the final concentration under the high-temperature condition of 65°C for 1 min, 2 min, 5 min, 15 min, 30 min, and 60 min respectively.

[0118] 1.3 Optimization results of NaOH termination temperature and termination time

[0119] After the reaction is completed, detect by SDS-PAGE. Among them, the SDS-PAGE detection includes the following steps:

[0120] Prepare a 20% SDS-PAGE separating gel. After the gel solidifies, it can be placed in the electrophoresis tank for electrophoresis. The specific formula of the separating gel is shown in Table 9.

[0121] Table 9

[0122]

[0123]

[0124] Take the products after different digestions, add 2×Loading buffer and mix well. Load 5 μL. Add 2×Loading buffer to the control group oligonucleotide and mix well, and also load 5 μL. Electrophorese at 220 V for 90 min, and take pictures with a gel imager.

[0125] The SDS-PAGE detection results are as Figure 3 shown. Treating with 50 mM NaOH at the final concentration under the condition of 65°C for 5 min has the best effect.

[0126] 2. Screening and Dosage Optimization of UDG Enzyme

[0127] Prepare the reaction system, and the specific formula is shown in Table 10, where 10×buffer is shown in Table 12.

[0128] Table 10

[0129]

[0130] Reaction conditions: Add U-1 / U-2 / U-3 / U-4 / U-5 / U-6 in Table 6 into the reaction system respectively, react at 37°C for 30 min, and treat with 50 mM NaOH at a final concentration at 65°C for 5 min. Among them, the concentrations of U-1 to U-2 and U-6 are diluted to 1 μg / μL; the original enzyme solution concentrations of U-3 to U-5 are low, and they are added according to the volume of the original solution.

[0131] The SDS-PAGE detection results are as Figure 4 shown. U-3 to U-5 have relatively high activities and can catalyze the formation of AP sites of 400 pmol oligonucleotides within 30 min of reaction time, and the reaction is basically complete. Subsequently, the activities of the three UDGs of U-3 to U-5 are further tested.

[0132] To distinguish the high and low activities of U-3 to U-5 enzymes, 1 / 2 / 5 μL of the enzyme diluted 20 times with the original solution are respectively included in a 50 μL reaction system and reacted at 37°C for 5 min. The SDS-PAGE results are as Figure 5 shown. U-3 and U-5 have relatively high activities compared to other UDG enzymes, and adding 1 μL can almost digest 200 pmol of the substrate. Since U-3 and U-5 belong to the same species, U-3 is subsequently used for experiments.

[0133] 3. Screening of U-3 Usage Concentration and Reaction Time

[0134] Prepare the reaction system, and the specific formula is shown in Table 11, where 10×buffer is shown in Table 12, and U-3 is added after being diluted 20 times with the original enzyme solution.

[0135] Table 11

[0136]

[0137] Reaction conditions: React at 37°C for 2 min, 5 min, 10 min, 15 min, and 30 min respectively, and treat with 50 mM NaOH at a final concentration at 65°C for 5 min.

[0138] The SDS-PAGE results are as Figure 6It is shown that U-3 with 5 μL of the original enzyme solution diluted 20 times can completely digest 200 pmol of the substrate under the condition of 37 °C for 2 min, and has a high enzyme activity, which is sufficient to meet the cleavage requirement of the substrate concentration. To save the enzyme amount, 1 μL of U-3 can be used to extend the reaction time to 5 min, which can meet the cleavage effect of the substrate concentration.

[0139] 4. Adjustment and Selection of Reaction Buffer

[0140] Prepare 3 kinds of reaction buffers, and the specific formulas are shown in Tables 12 to 14.

[0141] Table 12 10×UDG Reaction Buffer

[0142]

[0143] Table 13 10×APE Reaction Buffer

[0144]

[0145] Table 14 10× Modified Reaction Buffer

[0146]

[0147] Prepare the reaction system and conditions for the two-step method, and the specific formula is shown in Table 15.

[0148] Table 15

[0149]

[0150] Prepare the reaction system and conditions for the one-step method, and the specific formula is shown in Table 16.

[0151] Table 16

[0152]

[0153]

[0154] The SDS-PAGE results are as Figure 7 shown. To make the reaction simple and fast, the double-enzyme one-step method is selected for oligonucleotide cleavage reaction, and the modified buffer (Table 14) is superior to the UDG buffer (Table 12) and the APE buffer (Table 13).

[0155] 5. APE Enzyme Screening and Dosage Optimization

[0156] Prepare the reaction system and conditions for the two-step method, and the specific formula is shown in Table 17. Among them, the UDG concentration is diluted 20 times based on the original enzyme solution, and the APE enzyme is diluted to 0.5 μg / μL. After U-5 completes the primary reaction, then add A-1 to A-5, the modified buffer and water respectively, and the total system is 60 μL.

[0157] Table 17

[0158]

[0159] The results of SDS-PAGE are as Figure 8 shown. The enzyme activities of A-1, A-3, A-4, and A-5 are relatively high, among which the enzyme activity of A-3 is the highest. Subsequently, A-3 was used for experiments, and adding 1-8 μL of 0.5 μg / μL A-3 to the system could achieve a better cleavage effect.

[0160] 6. Optimization of DTT in the reaction buffer (one-step method)

[0161] In the components of the modified reaction buffer in Table 14, DTT was removed to prepare the modified buffer-2, and the specific formula is shown in Table 18. The one-step reaction system and conditions were used.

[0162] Table 18 10× Modified reaction buffer-2

[0163]

[0164] The detection results of SDS-PAGE are as Figure 9 shown. DTT has an inhibitory effect on the reaction, and the effect of the modified reaction buffer-2 is better than that of the modified reaction buffer, with more cleavage products. However, compared with the cleavage products obtained by NaOH lysis (the modified reaction buffer-2 + NaOH is used as the positive control: adding NaOH for lysis after one-step cleavage), the enzyme-catalyzed reaction in the modified reaction buffer-2 is still incomplete, and the system needs to be further optimized.

[0165] 7. Optimization of the MgCl2 concentration in the reaction buffer (one-step method)

[0166] Reaction systems with different concentrations of MgCl2 were prepared, and the specific formula is shown in Table 19. The one-step reaction system and conditions were used.

[0167] Table 19 10× Modified reaction buffer

[0168]

[0169] The detection results of SDS-PAGE are as Figure 10 shown. High concentrations of MgCl2 will inhibit the reaction, and the best effect is achieved when the MgCl2 concentration is 100 mM.

[0170] 8. Optimization of the KCl, Tris-HCl, PCNA, and BSA concentrations in the reaction buffer (one-step method)

[0171] Prepare reaction systems with different concentrations of KCl, Tris-HCl, PCNA, and BSA. The specific formulations are shown in Table 20. Use the one-step reaction system and conditions.

[0172] Table 20 10× Modified Reaction Buffer

[0173]

[0174] SDS-PAGE Results Figure 11 Show that BSA has a positive effect on the UDG reaction, but has no obvious effect on the one-step two-enzyme reaction; while PCNA has no obvious effect on the one-step two-enzyme reaction; high-concentration Tris-HCl inhibits both the UDG reaction and the one-step two-enzyme reaction, and the effect is best when the concentration of Tris-HCl is 200 mM; high-concentration KCl inhibits both the UDG reaction and the one-step two-enzyme reaction, and the effect is best when the concentration of KCl is 200 mM.

[0175] 9. Optimization of Reaction Temperature (One-Step Method)

[0176] Prepare the one-step reaction system and place it under different reaction temperature conditions. The specific formulations are shown in Tables 21 and 22.

[0177] Table 21

[0178]

[0179]

[0180] Table 22

[0181]

[0182] SDS-PAGE Results Figure 12 Show that high temperature inhibits the activity of UDG enzyme, and the activity of UDG enzyme is significantly weakened at 50°C; the optimal reaction temperature of A-3 is 37°C, and the optimal reaction temperature of A-5 is 50°C.

[0183] 10. Optimization of Reaction Time (One-Step Method)

[0184] Prepare the one-step reaction system and react it under different time conditions. The specific formulations are shown in Table 23. At the same time, the influence of the presence of PCNA in 10× buffer on the reaction effect was further verified.

[0185] Table 23

[0186]

[0187] SDS-PAGE Detection Results Figure 13It shows that the reaction effect is better without adding PCNA, and complete cleavage can be achieved within 5 minutes of reaction time.

[0188] In summary, A-3 and A-5 have relatively high enzyme activities in APE. After optimizing and comparing the temperature and time, A-3 has the highest enzyme activity. Two highly active enzymes, U-3 and A-3, are selected as the cleavage methods for solid-phase synthesis of DNA, and the reaction buffer formulation is finally confirmed as shown in Table 24.

[0189] Table 24 10× Reaction Buffer

[0190]

[0191] The enzyme types and dosages, enzyme reaction systems, and reaction conditions are finally confirmed as shown in Table 25.

[0192] Table 25

[0193]

[0194]

[0195] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0196] The above-described embodiments only represent several implementation manners of the present application. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. A buffer for nucleic acid cleavage, characterized in that, The buffer solution consists of components at the following working concentrations: 10 mM to 50 mM Tris-HCl, 10 mM to 100 mM KCl, 1 mM to 50 mM MgCl2, and water.

2. The buffer according to claim 1, characterized in that, The buffer solution consists of components at the following working concentrations: 10 mM to 30 mM Tris-HCl, 10 mM to 50 mM KCl, 5 mM to 20 mM MgCl2, and water; Optionally, the pH value of the Tris-HCl is 7.8 to 8.

8.

3. A kit for nucleic acid cleavage, characterized in that, The kit includes the buffer solution according to claim 1 or 2, UDG enzyme, and APE enzyme.

4. The kit according to claim 3, characterized in that, The UDG enzyme includes the UDG enzyme with a sequence shown in at least one of SEQ ID NO.1 to SEQ ID NO.6; Optionally, the UDG enzyme includes the UDG enzyme with a sequence shown in at least one of SEQ ID NO.3 to SEQ ID NO.

5.

5. The kit according to any one of claims 3 or 4, characterized in that, The APE enzyme includes the APE enzyme with a sequence shown in at least one of SEQ ID NO.7 to SEQ ID NO.11; Optionally, the APE enzyme includes the APE enzyme with a sequence shown in at least one of SEQ ID NO.7 and SEQ ID NO.9 to SEQ ID NO.

11.

6. A reaction system for nucleic acid cleavage, characterized in that, The reaction system includes the buffer solution, UDG enzyme, and APE enzyme in the kit according to any one of claims 3 to 5, nucleic acid, and water; Optionally, the nucleic acid includes ssDNA or solid-phase linked DNA, and uracil deoxynucleotide is incorporated at the pre-positioning site of the ssDNA or solid-phase linked DNA as the cleavage site; Optionally, every 50 μL of the reaction system includes 5 μL of 10× the buffer solution, 1 μL to 20 μL of the undiluted solution of the 20-fold diluted UDG enzyme, 0.5 μL to 8 μL of the APE enzyme at 0.4 μg / μL to 1 μg / μL, 100 pmol to 400 pmol of nucleic acid, and water.

7. Use of the buffer according to claim 1 or 2, or the kit according to any one of claims 3 to 5, or the reaction system according to claim 7 in the preparation of a product for cleaving nucleic acid from a solid phase.

8. A method for nucleic acid cleavage, characterized in that, The method includes performing cleavage using the buffer solution according to claim 1 or 2, the kit according to any one of claims 3 to 5, or the reaction system according to claim 6; Uracil deoxynucleotide is incorporated at the pre-positioning site of the nucleic acid as the cleavage site.

9. The method according to claim 8, characterized in that, The method for nucleic acid cleavage includes the method for cleaving the nucleic acid from the solid phase.

10. The method according to claim 9, characterized in that, The cleavage conditions include reacting at 37°C to 55°C for 2 min to 30 min, and then terminating the reaction with EDTA.

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