CN116426559A - Method for rapidly synthesizing target fragment from head by using long primer - Google Patents

Abstract

The invention discloses a method for quickly synthesizing a target fragment from scratch by using a long primer, which comprises the following steps: (1) Designing and synthesizing a plurality of long primers with overlapping bases and covering the whole target fragment; (2) Carrying out PCR amplification by using the even number of long primers obtained in the step (1) in an alternating overlapping manner to obtain a PCR product; (3) Directly integrating the PCR product obtained in the step (2) into a linearization vector by utilizing one-step homologous recombination to obtain a recombination product; (4) Transforming the recombinant product obtained in the step (3) into competent cells to obtain a transformant with the target gene; the method takes 2-5 hours from the design of the long primer in the target gene to the final product. The method provided by the invention is suitable for rapid synthesis of long fragment DNA, has the characteristics of short synthesis period and high accuracy, and is suitable for DNA synthesis with high G+C content, repeated sequence or complex secondary structure.

Description

A method for rapid de novo synthesis of target fragments using long primers

technical field

The invention belongs to the field of synthetic biology, and in particular relates to a method (Rapid DNA synthesis using long primers, RSLP) for rapidly de novo synthesizing a target fragment by using long primers.

Background technique

The chemical synthesis of DNA sequences provides a powerful tool for the efficient expression of genes in heterologous systems and the characterization of gene structure and function. However, since template DNA is not always readily available, chemical synthesis makes it possible to generate genes without the use of template DNA molecules. However, genetic chemical synthesis also has many disadvantages, including high price, long synthesis cycle, and difficulty in synthesizing DNA with high G+C content, repetitive sequence or complex secondary structure. Therefore, in order to avoid the experimental cycle becoming too long due to target DNA synthesis, it is very important to develop a simple, fast and economical DNA synthesis method.

In recent years, some studies have described some oligonucleotide-based DNA sequence synthesis and assembly methods, such as PCR-based thermodynamic equilibrium inside-out synthesis (TBIO), double-asymmetric PCR-based two-step gene synthesis (( DA-PCR), overlapping extension PCR (OE-PCR), PCR two-step synthetic DNA method (PTDS) and PCR-based precise synthesis (PAS), etc. However, there are obvious defects in these technologies, such as errors in PCR synthesis The rate is relatively high, the PCR system is redundant, and the PCR program is cumbersome.

Contents of the invention

Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for rapidly de novo synthesis of target fragments using long primers for the deficiencies of the prior art.

In order to solve the above technical problems, the present invention discloses a method for rapidly de novo synthesis of target fragments using long primers, that is, an improved version RSLP of the PTDS method for synthesizing long and accurate DNA sequences. Similar to other DNA PCR synthesis methods, the first step in the RSLP protocol is to design the DNA sequence to be synthesized; codons and G+C content can be optimized during this step, as well as poor restriction enzyme sites or secondary structures; according to DNA Sequence, design and chemically synthesize 80-100 nt oligonucleotides overlapping with adjacent oligonucleotides by about 18-25 bp, that is, long primers. Then in the second step, the aforementioned oligonucleotide set is used to generate small fragments of DNA with a length of about 500-1000 bp by PCR ( FIG. 1 ). Here, in order to save time, multiple sets of systems (synthesis of different fragments of the same gene) are simultaneously performed during the PCR step (choose the minimum Tm value as the annealing temperature). Among them, the two outermost primers in each group are used as forward/reverse outer primers, and the remaining largest even-numbered internal oligonucleotides are used as inner primers; high-fidelity DNA polymerase should be used for this step. Finally, in the third step, multiple groups of small fragments of DNA (500-1000 bp) were directly assembled into gene expression vectors for gene characterization by means of infusion one-step cloning (Figure 2).

The method for rapidly de novo synthesis of target fragments using long primers specifically includes the following steps:

(1) Design and synthesize multiple long primers with overlapping bases and covering the entire target fragment;

(2) Use the even-numbered long primers obtained in step (1) to carry out PCR amplification by alternate overlapping extension to obtain PCR products, that is, small fragments of DNA with a length of about 500 to 1000 bp;

(3) directly integrating the PCR product obtained in step (2) into a linearized vector by one-step homologous recombination to obtain a recombinant product;

(4) Transforming the recombinant product obtained in step (3) into a competent cell to obtain a transformant with the gene of interest;

The method takes 2-5 hours in total from the design of the long primer in the target gene to the final product, that is, steps (1)-(3).

In the above method, if annealing is involved, the temperature is above 45°C. For example, in the PCR process involved, the annealing temperature is above 45°C. In the multi-primer PCR program and the vector fragment linearization PCR program, the annealing temperature is 45°C. ℃ or more.

In step (1), select the appropriate number of primers according to the length of the designed target fragment, specifically, select a fragment of 80-100nt at the 5' end of the target gene as the forward outer primer, and select a fragment at the 3' end of the 80-100nt fragment Obtain an overlapping fragment of 18-25bp long, and then select a long primer fragment of 80-100nt from the (80-100-overlapping fragment) bp, and so on until the last primer design is completed; the 80-100nt, preferably 80-90nt, more preferably 90nt; the 18-25bp, preferably 18-22bp, more preferably 20bp.

In step (1), the overlap length of the base overlap is 18-25 bp, preferably 18-22 bp, more preferably 20 bp.

In step (1), the sequence length of the long primer is 80-100 nt, preferably 80-90 nt, more preferably 90 nt. An important parameter for DNA synthesis is the length of the oligonucleotides used for PCR. For the RSLP protocol, the present invention uses 90nt oligonucleotides because 90nt provides the best balance between error rate and production cost.

In step (2), the PCR adopts the template-free mode, and the outermost two primers of the even-numbered long primers obtained in step (1) are used as forward/reverse outer primers when overlapping and alternately extended, and the remaining long primers with the largest even number as an internal primer.

In step (2), the PCR system consists of 0.5 μl final concentration of 10 nM inner primer mixture, 1 μl final concentration of 0.2 μM forward outer primer, 1 μl final concentration of 0.2 μM reverse outer primer, 25 μl high-fidelity DNA polymerase A mixed system of enzyme, dNTP and buffer, made up to 50 μl of ddH ₂ O; preferably, the final concentration of 10 nM internal primer mixture is obtained by mixing 1 μl of internal primer with a concentration of 10 nM, that is, the internal primer All were obtained after equal volume mixing at a concentration of 10 nM.

Wherein, the mixed system of DNA polymerase and buffer is Vazyme P520-01, in some embodiments it can also be a high-fidelity enzyme, in some embodiments it can also use 2×Phanta Max Master Mix, which can make every 100,000bp <1 error. All previous gene synthesis methods have errors in the final product, mainly due to mutations in oligonucleotides and mutations introduced during DNA polymerase-mediated synthesis of the target DNA sequence.

In step (3), the linearized vector is obtained by using forward and reverse primers containing homology arms to linearize the vector by PCR technology.

Wherein, the vector after the linearization of the forward and reverse primers containing the homology arm has a 20bp homology arm sequence at the 5' end and the 3' end of the PCR product, respectively. The sequence is the same. If multiple fragments are inserted into the linearized vector together, the sequence at the 5' end of the linearized vector is the same as that of the large fragment composed of multiple small fragments, and the sequence at the 3' end is the same as that of the large fragment composed of multiple small fragments. The sequence at the 3' end is the same.

In step (3), the one-step homologous recombination system includes a linearized vector and n inserts, 5≥n≥1, such as n=1, n=2, n=3.

Wherein, the amount of the linearized vector and the n inserts is that the molar ratio of the linearized vector and the insert is 1:1, and the final concentration is 0.03 pmol of each linearized vector/insert used amount=[ 0.02 × linearized vector/insert base pairs] ng.

In step (3), the one-step homologous recombination has a recombination time of 30 minutes and a recombination temperature of 37°C; preferably, it is stored at 4°C after the recombination time is over.

It can be seen that the RSLP protocol provided by the present invention is relatively simple (multiple parallel PCRs are cloned onto the plasmid in one step), fast (from the design of the target gene to the final product ~4h), and accurate (≤1 error per 100,000bp synthesis) And the price is low.

Beneficial effect:

(1) Compared with the scheme similar to the experimental procedure of the present invention, the present invention selects the oligonucleotide chain (long primer) of 90nt for use, has not only shortened the synthesis period, reduced the difficulty of synthesis, but also saved economic cost, and the error rate Provides the best balance between and production costs.

(2) The present invention has adopted no template, utilizes the method for de novo DNA synthesis of long oligonucleotide chains (long primers), not only in terms of cost, is less than half of the de novo chemical synthesis of DNA chains currently on the market, but also in the synthesis of high GC It has irreplaceable advantages in terms of DNA chain content and complex structure. Most importantly, in terms of time consumption, the present invention greatly saves the time for users to acquire new genes. Under the specific gene fragment length, compared with the cycle of ordering genes in ordinary companies, the time consumption of the present invention to successfully construct the gene is 1/3 times, and with the increase of the gene fragment length, the time advantage of this invention will be eliminated Continue to expand, taking the synthesis of a 10kb DNA chain as an example, the time consumption of the present invention is 1/45 times that of chemical synthesis! It is very suitable for molecular biology laboratories without templates and urgently needing new genes.

(3) The present invention uses homologous recombination to combine multiple gene fragments. This method connects multiple DNA fragments with homologous arms, which can ensure the further improvement of the fidelity of the synthetic gene and save time. Compared with the overlap extension PCR method used by the traditional PAS method to connect multiple small fragments, the present invention directly adds 20bp homology arms to both ends of two adjacent DNA fragments at the beginning of primer design, and finally clones them in one step. Cloning of artificially synthesized genes into plasmid vectors. Despite the increasing fidelity of commercial DNA polymerases, there is no guarantee that the PCR process will be error-free, especially with regard to the principles of the present invention. In this reaction, the number of times that a certain number of enzymes bind the 3'-OH end of the primer is more than 5 times that of the template system. If the PTDS method of the prior art is used, it is very easy to synthesize large fragments using small fragments This is also one of the biggest pain points facing the PTDS law. The invention utilizes the one-step cloning technique of the homology arm to insert the synthesized fragment into any position of the vector, and is not limited by the presence or absence of restriction sites.

Description of drawings

The advantages of the above and/or other aspects of the present invention will become clearer as the present invention will be further described in detail in conjunction with the accompanying drawings and specific embodiments.

Figure 1 is a demonstration of the PCR process.

Figure 2 is a flow chart of the whole process.

Fig. 3 is a plasmid map of pET29a-AMP.

Figure 4 is a conceptual diagram of primer design.

Fig. 5 is the sequencing result (part) of Example 1.

Fig. 6 is the sequencing result (part) of Example 2.

Detailed ways

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

In the following examples, the sequence length of the designed and synthesized long primers is all 90 nt. Because in the market, taking Jinweizhi Company as an example, the primer synthesis fee standard is 90nt as the watershed, the primer synthesis fee is 4.2 yuan/nt for primers larger than 90nt, and the primer synthesis fee for 90nt or less is 0.45 yuan/nt. In terms of synthesis cycle and synthesis difficulty, the synthesis difficulty of primers larger than 90 nt is generally higher than that of primers smaller than or equal to 90 nt, and the synthesis cycle is also longer. In the synthesis of primers less than or equal to 90nt, taking the synthesis of a DNA sequence with a length of 860bp as an example, you can choose 12 primers with a length of 90nt, and the total length of multiple primers synthesized is 1080bp; you can also choose 21 primers with a length of 60nt. Primers, the total length of multiple synthetic primers is 1260bp, but the cost of the latter is 100 yuan higher than that of the former. Therefore, in the primer synthesis of the present invention, 90nt is selected as the sequence length of the long primer designed and synthesized.

The maximum time-consuming of PCR in the following examples 1 and 2 is 1.5h, that is, the PCR operation can be completed within 1.5h.

Embodiment 1: Construction of pET29a-EI-AMP plasmid

The target gene sequence EI [BBa_K1317003] was obtained, and its codon optimization was performed to make it suitable for Escherichia coli. The optimized sequence is shown in SEQ ID NO.3. Use the snapgene software to import the target gene and design the long primers used for synthesis. First, select a 90nt fragment at the 5' end of the target gene as a forward primer, obtain a 20bp long overlapping fragment at the 3' end of the 90nt fragment, and then select a 90nt long primer backward from the (90-overlapping fragment) bp Fragments, and so on until the last primer design is completed. Note: The extension direction of each designed primer must be opposite to that of the previous one. Ensure that the total number of primers designed is an even number (see Figure 4 for a visual display of primer design). The long primer sequences are shown in SEQ ID NO.4-NO.15. It takes 30 minutes.

Use the following steps to configure the system and perform PCR amplification reactions:

1. Multi-primer PCR: Synthesize a 855bp DNA strand. Time-consuming: 1h5min.

(1) The designed and synthesized 90nt long oligonucleotide primers were composed into a group, which contained 12 long oligonucleotide primers (inner primers: SEQ ID NO.5-14, forward outer primers: SEQ ID NO. 4. The reverse outer primer: SEQ ID NO.15) needs to synthesize a 855bp DNA fragment. Throughout the procedure, all reaction solutions should be stored and handled on ice unless otherwise stated.

(2) Mix 10 inner primers (except the outermost forward/reverse outer primers) for each group: take 1 μl (10 μM) of 10 inner primers and put them in a 200 μl PCR tube. Mix well by pipetting and centrifuge briefly to bring all liquid to the bottom of the tube.

(3) Take 0.5 μl of the inner primer mixture solution in step (2) and place it in a 200 μl PCR tube, and then take 1 μl (10 nM) of the forward outer primer and reverse outer primer respectively and place them in the above-mentioned 200 μl PCR tube. The final concentration of each internal primer was controlled at 10 nM, and the final concentration of the external primer was controlled at 0.2 μM. The multi-primer PCR system is shown in Table 1.

Table 1 Multi-primer PCR system setup

(4) Use the PCR program shown in Table 2

Table 2 Multi-primer PCR program settings

(5) 5 μl of the PCR product was added to 1.0% agarose gel containing 0.1% nucleic acid dye for electrophoresis for 30 min at a voltage of 125 V, using DL5000 DNA marker (MD102-01, Vazyme) as a reference.

(6) Plasmid pET29a-AMP is derived from laboratory preservation and synthesized by GenScript Biotechnology Co., Ltd. (Fig. 3), design the primer (forward primer nucleotide sequence as shown in SEQ ID NO.1, reverse primer nucleotide sequence as shown in SEQ ID NO.2) that contains homology arm, by the mode of PCR to make it linear. The PCR system and program are shown in Table 3 and Table 4.

Table 3 Vector fragment linearization PCR system

Table 4 Vector fragment linearization PCR program

2. Recover the DNA of the target band. Time-consuming: 15 minutes.

(1) Use the nucleic acid gel cutting equipment to cut off the target band, and use the FastPure Gel DNAExtraction Mini Kit Vazyme for gel recovery.

(2) Use an ultra-micro-volume ultraviolet spectrophotometer to quantify the products recovered from the gel (note that the concentration of DNA fragments recovered from the gel should be at least 20 ng/μl).

Three, one-step cloning. Time-consuming: 30min.

(1) Configure the one-step cloning system according to Table 5:

Table 5 One-step cloning system

Note: The molar ratio of linearized vector and insert is 1:1, linearized vector and insert quantification formula: the optimal amount of each linearized vector/insert with a final concentration of 0.03pmol=[0.02×linearized vector /number of base pairs of the insert] ng, which is very important for the success of the recombination; n=1.

(2) The recombination time was strictly controlled at 30 min, the recombination temperature was 37°C, and immediately stored at 4°C to obtain the recombination product pET29a-EI-AMP. The recombinant product can be stored at -20°C for one week, and can be thawed when needed.

4. Transformation of recombinant products

(1) Thaw chemically competent cells (DH5a Competent cell, Vazyme C502) for cloning on ice.

(2) Take 10 μl of the recombinant product and add it to 100 μl of competent cells, flick the tube wall to mix (do not vibrate to mix), and let stand on ice for 30 minutes.

(3) After heat-shocking in a water bath at 42°C for 45 sec, immediately place it on ice to cool for 2-3 min.

(4) Add 900 μl of SOC or LB medium (without adding antibiotics), and shake the bacteria at 37'C for 1 hour (rotating speed 200-250rpm).

(5) Preheat the LB plate solid medium containing kanamycin resistance at a final concentration of 100 mg/L in a 37° C. incubator.

(6) Centrifuge at 5,000 rpm (2400×g) for 5 minutes, and discard 900 μl of supernatant. Resuspend the bacteria with the remaining SOC or LB medium, and spread evenly on the plate containing the correct resistance with a sterile spreader stick.

(7) Incubate upside down in a 37°C incubator for 12-16 hours.

Five, verification

Pick the successfully transformed colonies, use colony PCR verification, inoculate the colony PCR positive transformants, expand culture, preserve, extract the plasmid, and carry out the PCR verification of the plasmid pET29a-EI-AMP. For transformants with positive verification results, entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to perform sequencing. If the sequencing result is correct, it is confirmed as the recombinant bacterium (see Figure 5 for the sequencing result).

Embodiment 2: Construction of pET29a-OVA plasmid

Obtain the target gene sequence OVA [BBa_K4150006], and optimize its codons to make it suitable for Escherichia coli. The optimized sequence is shown in SEQ ID NO.16. Use the snapgene software to import the target gene and design the long primers used for synthesis. First, a 90nt fragment is selected at the 5' end of the target gene as a forward primer, and a 20bp long overlapping fragment is obtained at the 3' end of the 90nt fragment. From (90 -overlapping fragment) bp and then select a 90nt long primer fragment backwards, and so on until the last primer design is completed. Note: The extension direction of each designed primer must be opposite to that of the previous one. Ensure that the total number of primers designed is an even number (see Figure 4 for a visual display of primer design). The long primer sequences are shown in SEQ ID NO.17-NO.34. It takes 40 minutes.

Use the following steps to configure the system and perform PCR amplification reactions:

1. Multi-primer PCR: synthesize two DNA strands with lengths of 571bp and 677bp respectively. Time-consuming: 1.5h.

(1) Divide all designed and synthesized 90nt oligonucleotide long primers into 2 groups of appropriate numbers, wherein the first group of 8 oligonucleotide primers (inner primers: SEQ ID NO.18-23, forward outer primers: SEQ ID NO.17, reverse outer primer: SEQ ID NO.24) need to synthesize a 571bp DNA fragment, the second group of 10 oligonucleotide primers (inner primer: SEQ ID NO.26-33, forward outer primer : SEQ ID NO.25, reverse outer primer: SEQ ID NO.34) need to synthesize a 677bp DNA fragment. Throughout the procedure, all reaction solutions should be stored and handled on ice unless otherwise stated.

(2) Premix the internal primers of each group: take two groups of 1 μl (10 μM) internal primers and place them in two 200 μl PCR tubes. Mix well by pipetting and centrifuge briefly to bring all liquid to the bottom of the tube.

(3) Take 0.5 μl of the two groups of internal primer mixture solutions in step (2) and place them in a 200 μl PCR tube, and then take 1 μl (10 nM) of each group’s corresponding forward and reverse external primers and place them in the above 200μl PCR tube. The final concentration of each internal primer was controlled at 10 nM, and the final concentration of the external primer was controlled at 0.2 μM. The multi-primer PCR system is shown in Table 6.

Table 6 Multi-primer PCR system setup

(4) Use the PCR program shown in Table 7

Table 7 Multi-primer PCR program setting

(5) 5 μl of PCR products were added to 1.0% agarose gel containing 0.1% nucleic acid dye for electrophoresis for 30 min at a voltage of 125 V, using DL5000 DNA marker (MD102-01, Vazyme) as a reference.

(6) Plasmid pET29a-AMP is derived from laboratory preservation and synthesized by GenScript Biotechnology Co., Ltd. (Fig. 3), design the primer (forward primer nucleotide sequence as shown in SEQ ID NO.35, reverse primer nucleotide sequence as shown in SEQ ID NO.36) that contains homology arm, by the mode of PCR to make it linear. The PCR system and program are shown in Table 8 and Table 9.

Table 8 Vector fragment linearization PCR system

Table 9 Vector fragment linearization PCR program

2. Recover the DNA of the target band. Time-consuming: 15 minutes.

(1) Use the nucleic acid gel cutting equipment to cut off the target band, and use the FastPure Gel DNAExtraction Mini Kit Vazyme for gel recovery.

(2) Use an ultra-micro-volume ultraviolet spectrophotometer to quantify the products recovered from the gel (note that the concentration of DNA fragments recovered from the gel should be at least 20 ng/μl).

Four, one-step cloning. Time-consuming: 30min.

(1) Configure the one-step cloning system according to Table 10:

Table 10 One-step cloning system

Note: The molar ratio of linearized vector and insert is 1:1, linearized vector and insert quantification formula: the optimal amount of each linearized vector/insert with a final concentration of 0.03pmol=[0.02×linearized vector /number of base pairs of the insert] ng, which is very important for the success of the recombination; n=2.

(2) The recombination time was strictly controlled at 30 minutes, the recombination temperature was 37°C, and immediately stored at 4°C to obtain the recombination product pET29a-OVA. The recombinant product can be stored at -20°C for one week, and can be thawed when needed.

4. Transformation of recombinant products

(1) Thaw chemically competent cells (DH5a Competent cell, Vazyme C502) for cloning on ice.

(2) Take 10 μl of the recombinant product and add it to 100 μl of competent cells, flick the tube wall to mix (do not vibrate to mix), and let stand on ice for 30 minutes.

(3) After heat-shocking in a water bath at 42°C for 45 sec, immediately place it on ice to cool for 2-3 min.

(4) Add 900 μl of SOC or LB medium (without adding antibiotics), and shake the bacteria at 37'C for 1 hour (rotating speed 200-250rpm).

(5) Preheat the LB plate solid medium containing kanamycin resistance at a final concentration of 100 mg/L in a 37° C. incubator.

(6) Centrifuge at 5,000 rpm (2400×g) for 5 minutes, and discard 900 μl of supernatant. Resuspend the bacteria with the remaining SOC or LB medium, and spread evenly on the plate containing the correct resistance with a sterile spreader stick.

(7) Incubate upside down in a 37°C incubator for 12-16 hours.

Five, verification

Pick the successfully transformed colonies, use colony PCR verification, inoculate the colony PCR positive transformants, expand culture, preserve, extract the plasmid, and carry out the PCR verification of the plasmid pET29a-OVA. For transformants with positive verification results, entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to perform sequencing. If the sequencing result is correct, it is confirmed as the recombinant bacterium (see Figure 6 for the sequencing result).

Integrating a complete set of experimental procedures, the improved method of de novo DNA synthesis using long primers in the present invention can synthesize rationally designed multiple DNA fragments with high efficiency and high fidelity. For conventional DNA synthesis, the time is reduced by at least 5 days.

The present invention provides an idea and method for using long primers to rapidly de novo synthesize target fragments. There are many methods and approaches to realize this technical solution. The above descriptions are only preferred embodiments of the present invention. Those of ordinary skill in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components that are not specified in this embodiment can be realized by existing technologies.

Claims (10)

1. A method for rapidly de novo synthesis of target fragments utilizing long primers, characterized in that, comprising the steps: (1) designing and synthesizing long primers with overlapping bases and covering the entire target fragment; the sequence length of the long primers is 80-100 nt, preferably 80-90 nt, more preferably 90 nt; (2) Using the even-numbered long primers obtained in step (1) to alternately overlap and extend PCR amplification to obtain a PCR product; (3) directly integrating the PCR product obtained in step (2) into a linearized vector by one-step homologous recombination to obtain a recombinant product; (4) Transforming the recombinant product obtained in step (3) into a competent cell to obtain a transformant with the gene of interest; The steps (1) to (3) take a total time of 2 to 5 hours. 2. The method according to claim 1, characterized in that the annealing temperature in the method is above 45°C. 3. The method according to claim 1, characterized in that, in step (1), the overlapping length of the base overlap is 18-25 bp, preferably 18-22 bp, more preferably 20 bp. 4. according to the described method of claim 1, it is characterized in that, in step (2), described PCR adopts template-free mode, two primers of the outermost when overlapping alternate extension in the gained long primer of even number step (1) are used as Forward/reverse outer primers, the remaining long primers with the largest even number are used as inner primers. 5. The method according to claim 4, characterized in that, in step (2), the PCR system consists of 0.5 μl final concentration of 10 nM inner primer mixture, 1 μl final concentration of 0.2 μM forward outer primer, and 1 μl final concentration. The concentration is a mixed system of 0.2 μM reverse outer primer, 25 μl high-fidelity DNA polymerase, dNTP and buffer, and ddH ₂ O is supplemented to 50 μl; preferably, the final concentration is 10 nM inner primer mixture is the inner The primers were obtained after equal volume mixing at a concentration of 10 nM. 6. The method according to claim 1, characterized in that, in step (3), the linearized vector is obtained by using primers containing homology arms to linearize the vector by PCR technology. 7. The method according to claim 1, characterized in that, in step (3), the one-step homologous recombination system includes a linearized vector and n inserts, 5≥n≥1. 8. The method according to claim 7, characterized in that, the molar ratio of the linearized carrier and the insert is 1:1, and the final concentration is 0.03pmol of each linearized carrier/insert usage=[0.02 × linearized vector/insert base pairs] ng. 9. The method according to claim 1, characterized in that, in step (3), the one-step homologous recombination has a recombination time of 30 minutes and a recombination temperature of 37°C. 10. The method according to claim 1, characterized in that, in step (3), after the recombination time is over, it is stored at 4°C.

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