Rapid plasmid assembly method suitable for high-throughput screening
By optimizing primer design and operating procedures, and employing dual homologous arm primers and high-throughput 96-well plate technology, the problems of low efficiency and poor specificity in high-throughput screening of traditional plasmid construction methods have been solved, and rapid and reliable plasmid assembly has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN MIAOLING BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-03-17
- Publication Date
- 2026-06-30
AI Technical Summary
Traditional plasmid construction methods are cumbersome, have low assembly efficiency and poor specificity in high-throughput screening, making it difficult to meet the needs of high-throughput screening.
By employing a dual-homological-arm primer design, optimizing the PCR amplification system and procedure, using compatible dual-enzyme buffer, thermophilic homologous recombinase, and high-throughput 96-well plate operation, combined with a dual verification method, we achieved efficient and rapid plasmid assembly.
It significantly improved the specificity and efficiency of PCR amplification, shortened the enzyme digestion reaction time, increased the recombination speed, met the needs of high-throughput screening, reduced the false positive rate, and improved the reliability of experiments.
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Figure CN122303277A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology experimental technology, specifically a rapid plasmid assembly method suitable for high-throughput screening. Background Technology
[0002] Plasmids, as core vectors for gene cloning and expression, play an irreplaceable role in molecular biology research, gene therapy, and biopharmaceuticals. With the development of high-throughput sequencing and functional genomics, higher demands are placed on the efficiency, throughput, and specificity of plasmid assembly. Traditional plasmid construction methods, such as enzyme digestion and ligation and homologous recombination, suffer from cumbersome procedures, low assembly efficiency, poor specificity, and difficulty in meeting the needs of high-throughput screening.
[0003] Homologous recombination, a commonly used plasmid construction technique, offers high flexibility, but it still faces numerous challenges in practical applications: target fragment amplification failure, low enzyme digestion efficiency, insufficient homologous recombination efficiency, low transformation efficiency, and difficulties in screening and verification. These problems are further amplified in high-throughput experiments, leading to longer experimental cycles and increased costs, severely hindering the progress of large-scale gene function screening. Therefore, developing an efficient, rapid, and highly specific plasmid assembly method suitable for high-throughput screening is of significant practical importance.
[0004] Based on this, a rapid plasmid assembly method suitable for high-throughput screening was designed. Summary of the Invention
[0005] In view of the above situation and to overcome the shortcomings of the prior art, the present invention provides a rapid plasmid assembly method suitable for high-throughput screening, which effectively solves the problems raised in the background art.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a rapid plasmid assembly method suitable for high-throughput screening, comprising the following steps:
[0007] Step 1, Design and preparation of targeted primers:
[0008] Design primer pairs containing two homologous arms, wherein the 5' homologous arm of the primer pair is 20-25 bp in length, the 3' specific binding fragment is 18-22 bp in length, the primer Tm value is 58-62℃, and the difference in Tm value between the upstream and downstream primers is ≤1℃; the primers are synthesized by solid-phase synthesis, purified by PAGE, and prepared into a 10 μmol / L primer stock solution with TE buffer and stored at -20℃;
[0009] Step 2: High-fidelity PCR amplification of the target fragment:
[0010] A 20 μL amplification system was constructed in a 96-well PCR plate, comprising: 7 μL sterile water, 10 μL 2× high-fidelity enzyme mixture, 1 μL upstream primer, 1 μL downstream primer, and 1 μL template DNA. The PCR plate was placed in a PCR instrument, and the program was set as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 1 min / kb, for a total of 30 cycles; final extension at 72℃ for 7 min, followed by incubation at 4℃. The amplified products were detected by 1.5% agarose gel electrophoresis, and the size of the target fragment was as expected.
[0011] Step 3: Double enzyme digestion of the vector and the target fragment:
[0012] A 50 μL restriction enzyme system was constructed in a 96-well enzyme digestion plate. The system consisted of: 41 μL sterile water, 5 μL 10×CutBuffer, 1 μg vector DNA, 1.5 μL EcoRI restriction enzyme, and 1.5 μL BamHI restriction enzyme. The target fragment digestion system had the same components as the vector digestion system, except that the vector DNA was replaced with the PCR purified product. The enzyme digestion plate was placed in a 37°C water bath for 25 min. After the reaction, 5 μL 10×Loading Buffer was added to terminate the reaction.
[0013] Step 4: Efficient homologous recombination ligation:
[0014] A 10 μL recombinant system was constructed using a 96-well plate. The system consisted of: 2 μL of 5× recombinase buffer, 1 μL of recombinase, 2 μL of digested vector, 4 μL of digested target fragment, and 1 μL of sterile water. The plate was placed in a 50℃ constant temperature metal bath for 20 min, and immediately placed on ice for 5 min after the reaction.
[0015] Step 5: High-throughput conversion and screening:
[0016] Remove competent cells from the -80℃ freezer, thaw on ice, add 100 μL of competent cells to each well of a 96-well cell culture plate, then add 5 μL of recombinant product, and incubate on ice for 30 min; heat shock at 42℃ for 90 s, then immediately incubate on ice for 2 min; add 900 μL of antibiotic-free LB medium to each well, and culture at 37℃ and 180 rpm on a shaker for 40 min; spread 100 μL of culture onto LB solid medium plates containing the corresponding antibiotic, and incubate upside down at 37℃ for 12 h;
[0017] Step 6: Recombinant plasmid verification: Single colony plasmids were extracted using a high-throughput plasmid extraction kit. The correctness of the recombinant plasmid was confirmed by PCR and sequencing verification. The PCR verification system was 20 μL, including: 14 μL sterile water, 10 μL 2×Taq enzyme mixture, 0.5 μL each of the verification primers, and 1 μL plasmid template. The amplification program was 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 25 cycles, with a final extension of 72℃ for 5 min. Sequencing verification used bidirectional sequencing. A sequence homology of ≥99.9% was considered a positive recombinant plasmid.
[0018] Preferably, the dual homologous arms in step 1 include a vector homologous arm and a target fragment homologous arm, wherein the vector homologous arm is completely complementary to the linearized vector terminal sequence, and the target fragment homologous arm is completely complementary to the target fragment end sequences.
[0019] Preferably, the 2× high-fidelity enzyme mixture in step 2 contains hot-start high-fidelity DNA polymerase, dNTPs, and Mg. 2+ And stabilizers, wherein the concentration of dNTPs is 2 mmol / L, Mg 2+ The concentration is 3 mmol / L.
[0020] Preferably, the 10×Cut Buffer in step 3 is a double enzyme digestion buffer compatible with EcoRI and BamHI, containing 500 mmol / L Tris-HCl, 100 mmol / L MgCl2, 1 mol / L NaCl, and a pH of 7.5.
[0021] Preferably, the recombinase in step 4 is a thermophilic homologous recombinase with an enzyme activity ≥5000 U / mL, and 0.5 μL of DNA polymerase inhibitor, EDTA, with a concentration of 10 mmol / L, is also added to the recombinant system.
[0022] Preferably, the competent cells in step 5 are DH5α competent cells with a transformation efficiency ≥10%. 8 cfu / μg DNA; the antibiotic concentration in the LB solid medium is 50 μg / mL Ampicillin or 100 μg / mL Kanamycin.
[0023] Preferably, the high-throughput plasmid extraction kit in step 6 uses the silica membrane adsorption method, with an extraction time ≤30min / 96 wells, a plasmid purity of A260 / A280 of 1.8-2.0, and a concentration ≥50ng / μL.
[0024] Compared with the prior art, the beneficial effects of the present invention are:
[0025] 1. This invention significantly improves the specificity and efficiency of PCR amplification by optimizing primer design, adopting a double homologous arm structure, and strictly controlling the primer Tm value, thus avoiding the problem of target fragment amplification failure.
[0026] 2. The PCR amplification system and procedure were optimized, and a hot-start high-fidelity enzyme mixture was used to reduce non-specific amplification and base mismatches, ensuring the integrity and accuracy of the target fragment;
[0027] 3. The use of a buffer compatible with both enzymes shortens the enzyme digestion reaction time, simultaneously processes the vector and the target fragment, and enables direct recombination of the enzyme digestion products, simplifying the operation process;
[0028] 4. The homologous recombination system was optimized by adding a DNA polymerase inhibitor, which improved the specificity and efficiency of the recombination reaction, shortened the recombination time to 20 minutes, and significantly improved the assembly speed.
[0029] 5. It integrates the high-throughput operation workflow of 96-well plates, realizing high-throughput from PCR amplification, enzyme digestion, recombination to transformation and screening, and can process a large number of samples at the same time to meet the needs of high-throughput screening.
[0030] 6. The use of a dual verification method ensured the correctness of the recombinant plasmid, reduced the false positive rate, and improved the reliability of the experiment. Attached Figure Description
[0031] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.
[0032] In the attached diagram:
[0033] Figure 1 This is a schematic diagram of the overall process of the method of the present invention; Detailed Implementation
[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0035] Depend on Figure 1 This invention relates to a rapid plasmid assembly method suitable for high-throughput screening, comprising the following steps:
[0036] Step 1: Design and preparation of targeted primers
[0037] Design primer pairs containing double homologous arms. The 5'-ends of the primer pairs are provided with double homologous arms, where the vector homologous arm has a length of 20 - 25 bp and is completely complementary to the terminal sequence of the linearized vector; the target fragment homologous arm has a length of 20 - 25 bp and is completely complementary to the sequences at both ends of the target fragment; the 3'-ends of the primers are specific binding fragments with a length of 18 - 22 bp to ensure specific binding to the template DNA. Predict the Tm values of the primers through software, control them within the range of 58 - 62 °C, and the difference in Tm values between the upstream and downstream primers is ≤1 °C to avoid inconsistent amplification efficiency caused by different annealing temperatures.
[0038] Synthesize primers using the solid-phase synthesis method, and purify them by polyacrylamide gel electrophoresis (PAGE) to remove impurities and short fragments during the synthesis process and improve the primer purity. The purified primers are formulated into a 10 μmol / L primer stock solution with TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH 8.0), aliquoted, and stored sealed in a -20 °C refrigerator to avoid primer degradation caused by repeated freezing and thawing.
[0039] Step 2: High-fidelity PCR amplification of the target fragment
[0040] Perform high-throughput amplification in a 96-well PCR plate. Construct a 20 μL amplification system for each well, and the components of the system are accurately proportioned as follows: 7 μL of sterile water, 10 μL of 2× high-fidelity enzyme mixture, 1 μL of upstream primer, 1 μL of downstream primer, and 1 μL of template DNA. Among them, the 2× high-fidelity enzyme mixture contains a hot-start high-fidelity DNA polymerase, dNTPs, Mg 2+ and stabilizers. The concentration of dNTPs is 2 mmol / L, and the concentration of Mg 2+ is 3 mmol / L, which can effectively reduce non-specific amplification and base mismatches.
[0041] Seal the loaded 96-well PCR plate and place it in a PCR instrument. Set the optimized amplification program: pre-denature at 94 °C for 5 min to completely denature the template DNA; then enter 30 cycles, each cycle including denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min / kb (the extension time is adjusted proportionally according to the length of the target fragment); after the cycle ends, perform a final extension at 72 °C for 7 min to ensure complete extension of the target fragment; finally, keep it at 4 °C to prevent product degradation.
[0042] After the amplification is completed, take 2 μL of the PCR product and mix it with 1 μL of 10× Loading Buffer, and perform 1.5% agarose gel electrophoresis for detection at a voltage of 120 V for 25 min. Observe through a gel imaging system. If the size of the target fragment is consistent with the expectation and there are no miscellaneous bands, it is qualified.
[0043] Step 3: Double digestion of the vector and the target fragment
[0044] The vector and target fragment were processed simultaneously in a 96-well enzyme digestion plate. Each well contained a 50 μL digestion system, consisting of: 41 μL sterile water, 5 μL 10×Cut Buffer, 1 μg vector DNA (or 2 μg PCR purified product), 1.5 μL EcoRI restriction enzyme, and 1.5 μL BamHI restriction enzyme. The 10×Cut Buffer was a dual digestion buffer compatible with both EcoRI and BamHI, containing 500 mmol / L Tris-HCl, 100 mmol / L MgCl2, and 1 mol / L NaCl, with a pH of 7.5, ensuring optimal activity of both enzymes.
[0045] Place the enzyme digestion plate in a 37°C water bath for 25 minutes, precisely controlling the reaction time to avoid over- or incomplete digestion. Immediately after the reaction, add 5 μL of 10× Loading Buffer to each well to terminate the reaction and prevent further digestion that could lead to fragment degradation. The digestion products can be used directly for subsequent recombinant reactions without additional purification, simplifying the process.
[0046] Step 4: Efficient homologous recombination ligation
[0047] High-throughput recombination reactions were performed using 96-well ligation plates. Each well contained a 10 μL recombination system, consisting of: 2 μL 5× recombinase buffer, 1 μL recombinase, 2 μL digested vector, 4 μL digested target fragment, and 1 μL sterile water. The recombinase was a thermophilic homologous recombinase with an activity ≥5000 U / mL, capable of efficiently catalyzing homologous recombination reactions at high temperatures. 0.5 μL of EDTA (10 mmol / L) was added to the recombination system as a DNA polymerase inhibitor to prevent non-specific ligation caused by residual high-fidelity enzyme.
[0048] After sealing the connecting plate, place it in a 50°C constant temperature metal bath for 20 minutes to ensure that the homologous arms are fully complementary and combined. After the reaction is completed, immediately transfer the connecting plate to ice to cool for 5 minutes to terminate the recombination reaction, stabilize the structure of the recombination product, and prevent its degradation or dechaining.
[0049] Step 5: High-throughput conversion and screening
[0050] Remove DH5α competent cells (transformation efficiency ≥10) from a -80℃ freezer. 8 Thaw the cells (cfu / μg DNA) on ice for 10 min to avoid decreased competent cell viability due to room temperature storage. Add 100 μL of competent cells to each well of a 96-well cell culture plate, then add 5 μL of recombinant product, gently pipette to mix, and incubate on ice for 30 min to allow the recombinant plasmid to fully bind to the competent cells.
[0051] The cell culture plate was heat-shocked in a 42°C water bath for 90 seconds, with precise control of the heat shock time and temperature to promote plasmid entry into the cells. After heat shock, the culture plate was immediately transferred to ice and cooled for 2 minutes to allow the competent cell membranes to stabilize. 900 μL of antibiotic-free LB medium was added to each well, and the culture plate was incubated in a shaker at 37°C and 180 rpm for 40 minutes to allow the cells to recover and express antibiotic resistance genes.
[0052] Take 100 μL of culture and spread it evenly on an LB agar plate containing the corresponding antibiotic (50 μg / mL Ampicillin or 100 μg / mL Kanamycin), with one sample per plate. Invert the plates and incubate them in a 37°C incubator for 12 hours to prevent condensation from causing colony spread and to ensure single colony formation.
[0053] Step Six: Recombinant Plasmid Validation
[0054] Single colony plasmids were extracted using a high-throughput plasmid extraction kit (silica membrane adsorption method). This kit can complete plasmid extraction in 96 wells within 30 minutes. The extracted plasmids had an A260 / A280 purity of 1.8-2.0 and a concentration of ≥50 ng / μL, which met the requirements for subsequent validation.
[0055] The correctness of the recombinant plasmid was confirmed by PCR and sequencing. The PCR verification system consisted of 20 μL, including: 14 μL sterile water, 10 μL 2×Taq enzyme mixture, 0.5 μL each of the verification primers, and 1 μL plasmid template. The amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 25 cycles; and a final extension at 72℃ for 5 min. The amplified products were detected by agarose gel electrophoresis; the appearance of a band of the expected size indicated a positive PCR result.
[0056] Bidirectional sequencing was performed on PCR-positive samples. The sequencing results were compared with the designed sequence. If the homology was ≥99.9%, the positive recombinant plasmid was qualified and could be used for subsequent high-throughput screening experiments.
Claims
1. A rapid plasmid assembly method suitable for high-throughput screening, comprising the following steps: Step 1, Design and preparation of targeted primers: Design primer pairs containing two homologous arms, wherein the 5' homologous arm of the primer pair is 20-25 bp in length, the 3' specific binding fragment is 18-22 bp in length, the primer Tm value is 58-62℃, and the difference in Tm value between the upstream and downstream primers is ≤1℃; the primers are synthesized by solid-phase synthesis, purified by PAGE, and prepared into a 10 μmol / L primer stock solution with TE buffer and stored at -20℃; Step 2: High-fidelity PCR amplification of the target fragment: A 20 μL amplification system was constructed in a 96-well PCR plate, comprising: 7 μL sterile water, 10 μL 2× high-fidelity enzyme mixture, 1 μL upstream primer, 1 μL downstream primer, and 1 μL template DNA. The PCR plate was placed in a PCR instrument, and the program was set as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 1 min / kb, for a total of 30 cycles; final extension at 72℃ for 7 min, followed by incubation at 4℃. The amplified products were detected by 1.5% agarose gel electrophoresis, and the size of the target fragment was as expected. Step 3: Double digestion of the vector and the target fragment: A 50 μL restriction enzyme system was constructed in a 96-well enzyme digestion plate. The system consisted of: 41 μL sterile water, 5 μL 10×Cut Buffer, 1 μg vector DNA, 1.5 μL EcoRI restriction enzyme, and 1.5 μL BamHI restriction enzyme. The target fragment digestion system had the same components as the vector digestion system, except that the vector DNA was replaced with the PCR purified product. The enzyme digestion plate was placed in a 37°C water bath for 25 min. After the reaction, 5 μL of 10×Loading Buffer was added to terminate the reaction. Step 4: Efficient homologous recombination ligation: A 10 μL recombinant system was constructed using a 96-well plate. The system consisted of: 2 μL of 5× recombinase buffer, 1 μL of recombinase, 2 μL of digested vector, 4 μL of digested target fragment, and 1 μL of sterile water. The plate was placed in a 50℃ constant temperature metal bath for 20 min, and immediately placed on ice for 5 min after the reaction. Step 5: High-throughput conversion and screening: Remove competent cells from the -80℃ freezer, thaw on ice, add 100 μL of competent cells to each well of a 96-well cell culture plate, then add 5 μL of recombinant product, and incubate on ice for 30 min; heat shock at 42℃ for 90 s, then immediately incubate on ice for 2 min; add 900 μL of antibiotic-free LB medium to each well, and culture at 37℃ and 180 rpm on a shaker for 40 min; spread 100 μL of culture onto LB solid medium plates containing the corresponding antibiotic, and incubate upside down at 37℃ for 12 h; Step 6: Recombinant plasmid verification: Single colony plasmids were extracted using a high-throughput plasmid extraction kit. The correctness of the recombinant plasmid was confirmed by PCR and sequencing verification. The PCR verification system was 20 μL, including: 14 μL sterile water, 10 μL 2×Taq enzyme mixture, 0.5 μL each of the verification primers, and 1 μL plasmid template. The amplification program was 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 25 cycles, with a final extension of 72℃ for 5 min. Sequencing verification used bidirectional sequencing. A sequence homology of ≥99.9% was considered a positive recombinant plasmid.
2. The rapid plasmid assembly method for high-throughput screening according to claim 1, characterized in that: The dual homologous arms mentioned in step 1 include a vector homologous arm and a target fragment homologous arm. The vector homologous arm is completely complementary to the linearized vector terminal sequence, and the target fragment homologous arm is completely complementary to the target fragment end sequences.
3. The rapid plasmid assembly method for high-throughput screening according to claim 1, characterized in that: The 2× high-fidelity enzyme mixture mentioned in step 2 contains hot-start high-fidelity DNA polymerase, dNTPs, and Mg. 2+ And stabilizers, wherein the concentration of dNTPs is 2 mmol / L, Mg 2+ The concentration is 3 mmol / L.
4. The rapid plasmid assembly method for high-throughput screening according to claim 1, characterized in that: The 10×Cut Buffer mentioned in step 3 is a double enzyme digestion buffer compatible with EcoRI and BamHI, containing 500 mmol / L Tris-HCl, 100 mmol / L MgCl2, 1 mol / L NaCl, and a pH of 7.
5.
5. The rapid plasmid assembly method for high-throughput screening according to claim 1, characterized in that: The recombinase mentioned in step 4 is a thermophilic homologous recombinase with an enzyme activity ≥5000 U / mL. 0.5 μL of DNA polymerase inhibitor, EDTA, with a concentration of 10 mmol / L, is also added to the recombinant system.
6. The rapid plasmid assembly method for high-throughput screening according to claim 1, characterized in that: The competent cells mentioned in step 5 are DH5α competent cells with a transformation efficiency ≥10%. 8 cfu / μg DNA; the antibiotic concentration in the LB solid medium is 50 μg / mL Ampicillin or 100 μg / mL Kanamycin.
7. The rapid plasmid assembly method for high-throughput screening according to claim 1, characterized in that: The high-throughput plasmid extraction kit described in step 6 uses the silica membrane adsorption method, with an extraction time of ≤30 min / 96 wells, a plasmid purity of A260 / A280 of 1.8-2.0, and a concentration of ≥50 ng / μL.