CN104212827B - It is independent of the rapid molecular cloning process of bioengineered enzyme - Google Patents

Abstract

The invention belongs to the field of molecular biology, and in particular relates to a rapid molecular cloning method independent of bioengineering enzymes. This fast molecular cloning method not relying on bioengineering enzymes comprises the following steps: a, primer design and synthesis; respectively design and synthesize two pairs of primers, the first pair is the forward primer IF of the target gene and the reverse primer IR of the target gene, the second The pair is the carrier forward primer VF and the carrier reverse primer VR; b. Perform PCR to obtain the linear target gene and linear target carrier DNA respectively; c. Mix and place the two PCR products to make the linear target gene and linear target carrier DNA spontaneously Connect to form a circle to obtain a vector containing the target gene. Compared with the traditional molecular cloning technology, the fast molecular cloning method independent of bioengineering enzyme has the advantages of high speed, high efficiency, low cost, low random error and the like. have a broad vision of application.

Description

A Rapid Molecular Cloning Method Independent of Bioengineered Enzymes

technical field

The invention belongs to the field of molecular biology, and in particular relates to a rapid molecular cloning method independent of bioengineering enzymes.

Background technique

Following the completion of the human whole genome sequence map, the whole genome sequence maps of multiple species have also been completed one after another. The primary structure information of gene sequences provides an information basis for human beings to explore the structure and function of individual genes and related proteins. As a result, human beings have entered the era of post-genome research. However, in the face of massive gene sequence information, traditional molecular cloning technology is inefficient, costly, and has large random errors, which can no longer meet the needs of scientific research. The bottleneck of molecular cloning technology limits the comprehensive and in-depth analysis and Research. With the completion of whole-genome sequencing of more species and the discovery of more new species, post-genome research urgently needs a new high-throughput molecular cloning technology.

On the basis of traditional molecular cloning methods, many new gene cloning methods have emerged, including the use of new technologies such as homologous recombination sequences, specific linkers, and fast ligases. However, the establishment of all these new methods requires the use of corresponding bioengineering enzymes, such as various recombinases, T4 ligases, and restriction endonucleases. The use of these bioengineered enzymes increases the complexity of the experimental process and also increases the cost of the experiment. In order to solve the bottleneck problems such as complicated molecular cloning experiment process, high cost, and difficult quality control, new breakthroughs in molecular cloning methodology are needed in this field.

Contents of the invention

The technical problem to be solved by the present invention is that various bioengineering enzymes are required for molecular cloning in this field, which leads to complicated experimental procedures and high costs.

The technical solution of the present invention to solve the above technical problems is to provide a rapid molecular cloning method that does not rely on bioengineering enzymes.

This rapid molecular cloning method independent of bioengineering enzymes comprises the following steps:

a. Primer design and synthesis; design and synthesize two pairs of primers respectively, the first pair is the target gene forward primer IF and the target gene reverse primer IR, and the second pair is the vector forward primer VF and vector reverse primer VR;

b. Perform PCR to obtain linear target gene and linear target carrier DNA respectively;

c. The two PCR products are mixed and placed, so that the linear target gene and the linear target carrier DNA are spontaneously connected into a circular shape to obtain a carrier containing the target gene.

Further, the above method also includes step d: transforming bacteria with the product obtained in step c.

Further, the above method also includes step e: selecting a single clone colony to extract a plasmid, performing PCR verification and sequencing identification, and obtaining a recombinant vector containing the target gene.

Wherein, in the target gene forward primer IF and the target gene reverse primer IR described in step a of the above method, the target gene forward primer IF contains two parts, Overlap I1 primer and I2 primer, and the sequence of the Overlap I1 primer part and the insertion position of the target vector The sequence of 12-21 bases at the 5' end of the target gene is the same, and the sequence of the I2 primer part is the same as the sequence of 10-30 bases at the 5' end of the target gene; IR also contains two parts, namely Overlap I3 primer and I4 primer , The sequence of the Overlap I3 primer is reverse complementary to the sequence of 12-21 bases at the 3' end of the vector insertion position, and I4 is reverse complementary to the sequence of 10-30 bases at the 3' end of the target gene, where I1 and I3 bases The number is equal, and the number of bases is 12-21.

Preferably, the sequence of the I2 primer part of the target gene forward primer IF described in the above method step a is identical to the sequence of the first 20 bases at the 5' end of the target gene; The 20-base sequence is reverse complementary.

Wherein, the vector forward primer VF and the vector reverse primer VR described in step a of the above method are based on the 5' to 3' direction of the vector, and VF is the same as the 24-30 base sequence at the 3' end of the vector to be inserted, VR is reverse complementary to the 24-30 base sequence at the 5' end of the vector to be inserted.

The method for designing two pairs of primers of the present invention can be referred to in schematic diagram 10.

Wherein, the bacterium in the above method is Escherichia coli. DMT-competent Escherichia coli is preferred.

Wherein, the mixing and placing described in step C of the above method is carried out at room temperature, and the time is 30 to 90 minutes. Further, the mixing time is 30-60 minutes.

Wherein, the vector described in the above method is a plasmid.

Compared with the traditional molecular cloning technology, the fast molecular cloning method independent of bioengineering enzyme has the advantages of high speed, high efficiency, low cost, low random error and the like. Compared with the current rapid cloning method, it has more obvious advantages, that is, in the whole experimental process, there is no need to use any restriction endonuclease, ligase, recombinase system and other engineering enzymes, only DNA polymerase is needed. What is particularly valuable is the unique design method of the linker of this technology, which is not limited by the restriction enzyme cutting sites in commercial vectors, and can insert genes into any position of the plasmid, which can greatly expand the types of vectors used in molecular cloning, and can be used for vectors. any modification

In addition, the process of the method of the present invention is extremely simple, shortening the work of a skilled technician from three to four days to two days, and shortening the working time by 30% to 50%. The experimental cost can be reduced by up to 50%, and the success rate can reach 90%. have a broad vision of application.

Description of drawings

Figure 1. Reaction number A: EcoI-C domain, two PCRs to obtain linear target gene and linear target vector.

Figure 2. Plasmid was extracted from the monoclonal colony obtained by transforming Escherichia coli DMT with the vector obtained by picking reaction number A, and identified by PCR; plasimid1 is the result of plasmid extraction, and PCR of plasimid1 is the result of PCR verification of the plasmid.

Figure 3. Reaction number B: Pc109, linear target gene and linear target vector obtained by two PCRs respectively

Figure 4. The vector obtained by picking reaction number B was transformed into a monoclonal colony to extract a plasmid and identified by PCR; plasimid1 is the result of plasmid extraction, and PCR of plasimid1 is the result of PCR verification of the plasmid.

Figure 5. Reaction number C: BCRP1, target gene and linear target vector obtained by two PCRs respectively.

Figure 6. Plasmids extracted from monoclonal colonies obtained by transforming Escherichia coli DMT with the vector obtained by picking the linkers of 18bp and 21bp in reaction number C, PCR identification, BCRP118 and BCRP121PCR verification results are the verification results when the linkers are 18bp and 21bp respectively , it can be seen that the positive cloning rate can reach 75% when the linker is 18bp, and the positive cloning rate can reach 100% when the linker is 21bp.

Figure 7. Plasmids were extracted from monoclonal colonies obtained by transforming Escherichia coli DMT with the vector obtained by picking reaction number C with a linker of 12 bp. PCR identification, plasimid1 is the result of plasmid extraction, and PCR of plasimid1 is the result of PCR verification of the plasmid.

Figure 8. Plasmids were extracted from monoclonal colonies obtained by transforming Escherichia coli DMT with the vector obtained in reaction number C with a linker of 15 bp, identified by PCR, plasimid1 was the result of plasmid extraction, and PCR of plasimid1 was the result of PCR verification of the plasmid.

Figure 9. The experimental results of DMT competent cells and DH5α competent cells. DH5α competent cells must be used in conjunction with DpnI.

Figure 10. Schematic diagram of primer design principle.

Figure 11. Schematic diagram of the incubation connection principle.

detailed description

The inventive method can realize in the following manner specifically:

a. Primer design and synthesis; design and synthesize two pairs of primers respectively, the first pair is the target gene forward primer IF and the target gene reverse primer IR, and the second pair is the vector forward primer VF and vector reverse primer VR;

b. Perform PCR to obtain linear target gene and linear target carrier DNA respectively;

c. The two PCR products are mixed and placed, so that the linear target gene and the linear target carrier DNA are spontaneously connected into a circular shape to obtain a carrier containing the target gene.

Further, according to the requirements of cloning work, the method of the present invention may include step d or e. d: transforming bacteria with the product obtained in step c. e: Select single-clonal colonies to extract plasmids, perform PCR verification and sequencing identification, and obtain recombinant vectors containing the target gene.

The key point of the method of the present invention lies in step a. Refer to Fig. 10 and Fig. 11 for the method and working principle of the design of the two pairs of primers of the present invention.

Specifically, among the target gene forward primer IF and the target gene reverse primer IR described in step a, the target gene forward primer IF contains two parts, the Overlap I1 primer and the I2 primer, and the sequence of the Overlap I1 primer part is related to the insertion position of the target vector. The sequence of 12-21 bases at the 5' end of the target gene is the same, and the sequence of the I2 primer part is the same as the sequence of 10-30 bases at the 5' end of the target gene; IR also contains two parts, namely Overlap I3 primer and I4 primer , The sequence of the Overlap I3 primer is reverse complementary to the sequence of 12-21 bases at the 3' end of the vector insertion position, and I4 is reverse complementary to the sequence of 10-30 bases at the 3' end of the target gene, where I1 and I3 bases The number is equal, and the number of bases is 12-21.

Preferably, the sequence of the I2 primer part of the target gene forward primer IF described in the above method step a is identical to the sequence of the first 20 bases at the 5' end of the target gene; The 20-base sequence is reverse complementary.

Specifically, the vector forward primer VF and vector reverse primer VR described in step a of the above method are based on the 5' to 3' direction of the vector, VF and the 24-30 base sequence at the 3' end of the vector to be inserted Similarly, VR is reverse complementary to the 24-30 base sequence at the 5' end of the vector to be inserted.

Wherein, step b of the above method is to perform two PCRs respectively. Respectively, the target gene forward primer IF and the target gene reverse primer IR are amplified by PCR with the template of the target gene to obtain a linear target gene; the vector forward primer VF and the vector reverse primer VR are PCR-amplified with the linearized vector template Amplify the linearized target vector DNA. Obviously, the linearized vector template is matched with vector forward primer VF and vector reverse primer VR.

Wherein, the bacterium in the above method is Escherichia coli. DMT-competent Escherichia coli is preferred.

Wherein, the mixing and placing described in step C of the above method is carried out at room temperature, and the time is 30 to 90 minutes. Further, the mixing time is 30-60 minutes.

Wherein, the vector described in the above method is a plasmid.

Using the primer pair involved in the present invention to carry out PCR reaction, Overlap I1 and Overlap I3 will respectively form spontaneous connection with the target vector and assemble into a circular shape. This avoids the use of ligases in traditional cloning methods. Overlap is generated by primer design in the PCR reaction, which avoids the use of restriction enzymes in traditional cloning methods, and also avoids the recovery of linear vectors in the gel after enzyme digestion. In this way, the gene can be inserted into any position of the plasmid without the restriction of the restriction enzyme cutting site in the commercial vector.

In order to verify that the method of the present invention is applicable to various target genes with different lengths and base compositions, as well as applicable to different types of vectors, a series of verification experiments were carried out.

Embodiment 1 uses the method of the present invention to carry out molecular cloning

The primer sequences used in this example are shown in Table 1, and the information of each target gene is shown in Table 2. The vectors used are respectively plasmid vectors: PET28a, pTEV. Various reagents and carriers are commercially available. Both HS DNA Polymerase and DpnI restriction endonuclease were from Treasure Bioengineering (Dalian) Co., Ltd., and DMT competent cells were Transgen DMTChemically Competent Cells.

Table 1. Various primer sequences used in Example 1

Table 2. Information of the target gene used in Example 1

The specific process is:

1. According to the sequence of the disclosed carrier to be used, and the target gene fragment to be amplified in Table 2, design and synthesize various primers IF, IR and VF, VR required by the method of the present invention according to the method of the present invention, specifically See Table 1.

See Figure 10 for the design principle of primers

2. In each experiment, use the corresponding primers recorded in Table 2 to perform two PCRs on IF, IR, VF, and VR, respectively, to obtain linear target gene and linear target vector DNA fragments.

The two PCR reaction systems of each test are composed of the following components: 1) IF and IR (or VF and VR), 2) DNA template containing the target gene (or target vector), 3) dNTP, 4) high-fidelity DNA Polymerase PrimeSTAR, 5). PCR buffer used with PrimeSTAR, 6). Ultrapure water.

The reaction conditions were: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 s, annealing temperature (Tm-5)°C for 30 s, extension temperature at 72°C, duration set at 1000 bp/min; cycle number 18.

3. Two PCR products were mixed and incubated:

Reaction system: target gene 40ul~50ul+carrier linear DNA 40ul~50ul, temperature 37°C, time: 1h.

4. Take 2-3ul of the reaction product by conventional method to transform competent Escherichia coli DMT.

5. Pick single clone colonies to extract plasmids by conventional methods, carry out PCR verification and sequencing identification, and obtain Escherichia coli DMT which has been successfully transformed.

(1) Using the method of the present invention to clone gene fragments of different lengths

experiment procedure:

1. Use the primers provided in Table 1 to carry out the PCR reaction according to the design in Table 3.

The reaction condition of table 3 embodiment (one)

2. The products of each PCR were mixed and incubated separately according to the grouping in Table 4.

Table 4 Mixed Incubation Reaction Design

Mixed Incubation Reaction No. Reactant (the product of the corresponding PCR reaction number) A 1+2 B 1+3 C 1+4

Reaction system: target gene 40ul~50ul+carrier linear DNA 40ul~50ul, temperature 37°C, time: 1h.

3. The conventional method draws 2-3ul of the reaction product to transform competent E. coli DMT.

4. According to the conventional method, single-clonal colonies were selected according to the reaction sequence to extract plasmids, and PCR verification and sequencing identification were performed to obtain successfully transformed Escherichia coli DMT.

5. Experimental results:

Reaction No. A: The target gene is EcoI-C domain, see Figure 1 and Figure 2 for the results. Reaction No. B: The target gene is Pc109, see Figure 3 and Figure 4 for the results. Reaction No. C: The target gene is BCRP1, see Figure 5, Figure 6, Figure 7, and Figure 8 for the results.

The incubation reaction numbers A, B, and C reflect the ligation reaction of fragments of different lengths. The purpose of this experiment is to verify whether the target genes of different lengths have an impact on the ligation efficiency. From the experimental results, it can be concluded that the target genes of different lengths have an There is no significant difference in the efficiency of the proposed rapid cloning methods, and they all have high efficiency, which meets the requirements for the construction of recombinant vectors applied to target genes of different lengths.

(2): Comparison experiment between DMT competent cells and DH5α competent cells

1. Use the primers provided in Table 1 to carry out a PCR reaction according to the conditions designed in Table 5, and see Figure 9 for the PCR results.

The reaction condition of table 5 embodiment (two)

Each PCR product was mixed and incubated according to the design in Table 6.

Table 6 Mixed Incubation Reaction Design

Mixed Incubation Reaction No. Reactant (the product of the corresponding PCR reaction number) D. 5+6 E. Buffer for 5+6+DpnI+DpnI

Reaction system: target gene 40ul~50ul+carrier linear DNA 40ul~50ul, temperature 37°C, time: 1h.

2. Absorb and incubate 2-3 ul of the reaction product by conventional method respectively, transform the competent Escherichia coli DMT according to the reaction number D, and transform the competent cell DH5α according to the reaction number E.

3. According to the conventional method, single-clonal colonies were selected according to the reaction numbers D and E to extract plasmids, and PCR verification and sequencing identification were performed to obtain successfully transformed Escherichia coli DMT and DH5α. The results are shown in Figure 9.

4. Experimental results: DMT-competent Escherichia coli was used to directly remove methylated template plasmids. The reaction system consisted of two PCR product mixtures (target gene 40ul~50ul+ carrier linear DNA 40ul~50ul), and the reaction conditions were 37°C. Incubate for 1 h; DMT competent cells do not affect the cloning efficiency. Except for this experiment, the above and below clones were all carried out using DMT competent cells.

(3) Cloning with primer connectors of different lengths

1. Use the primers provided in Table 1 to carry out PCR reactions according to the design in Table 7.

The reaction condition of table 7 embodiment (three)

2. Each PCR product was mixed and incubated according to the design in Table 8.

Table 8 Mixed Incubation Reaction Design

Mixed Incubation Reaction No. Reactant (the product of the corresponding PCR reaction number) f 7+8 G 7+9 h 7+10 I 7+11

Reaction system: target gene 40ul~50ul+carrier linear DNA 40ul~50ul, temperature 37°C, time: 1h.

3. Conventional method: Pipette 2-3 ul reaction products of each of reaction sequences F, G, H, and I to transform competent Escherichia coli DMT.

4. According to the conventional method, single-clonal colonies were picked to extract plasmids according to the reaction sequence, and PCR verification and sequencing identification were performed to obtain E. coli DMT that was successfully transformed.

5. Experimental results

Experimental conclusion: when the Overlap is 12bp, the rapid ligation cloning method was used to ligate BCRP1 and pET28a, and the ligation was successful. See Figure 7 for the results.

Experimental conclusion: when the Overlap is 15bp, the method of the present invention is used to connect BCRP1 and pET28a, and the connection is successful. The results are shown in FIG. 6 .

When the Overlap is 18bp or 21bp, the method of the present invention is used to connect BCRP1 and pET28a, and the connection is successful. The result is shown in FIG. 8 .

In summary, Overlap can successfully carry out the ligation reaction between 12-21bp.

In general, this embodiment has verified that the method of the present invention can successfully insert DNA fragments of different sizes into different vectors; it has verified that the primer connectors of different lengths involved in the scope of the present invention can also be successfully cloned; it has also been verified that DMT is used to sense The actual effect of state cells replacing demethylase DpnI in rapid molecular cloning technology is satisfactory. In this example, after 18 cycles, the fidelity of DNA polymerase can still be guaranteed. Traditional PCR reactions often require 30 cycles, resulting in the amplification of non-specific products. Therefore, purification of PCR products is required to remove non-specific products. None of these are required in this method.

Claims (9)

1. The rapid molecular cloning method that does not rely on bioengineering enzyme is characterized in that comprising the following steps: a. Primer design and synthesis; design and synthesize two pairs of primers respectively, the first pair is the target gene forward primer IF and the target gene reverse primer IR, and the second pair is the vector forward primer VF and vector reverse primer VR; In the target gene forward primer IF and the target gene reverse primer IR, the target gene forward primer IF contains two parts: Overlap I1 primer and I2 primer. The sequence of the bases is the same, the sequence of the I2 primer part is the same as the sequence of 10-30 bases at the 5' end of the target gene; IR also contains two parts, namely the Overlap I3 primer and the I4 primer, the sequence of the Overlap I3 primer and the vector The sequence of 12-21 bases at the 3' end of the insertion position is reverse complementary, and I4 is reverse complementary to the sequence of 10-30 bases at the 3' end of the target gene; The vector forward primer VF and the vector reverse primer VR are based on the 5' to 3' direction of the vector. VF is the same as the 24-30 base sequence at the 3' end of the vector to be inserted, and VR is 5' to the vector to be inserted. 24-30 base sequences at the end are reverse-complementary; b. Perform PCR to obtain linear target gene and linear target carrier DNA respectively; c. The two PCR products are mixed and placed, so that the linear target gene and the linear target carrier DNA are spontaneously connected into a circular shape to obtain a carrier containing the target gene. 2. The method according to claim 1, further comprising step d: transforming bacteria with the product obtained in step c. 3. The method according to claim 2, further comprising step e: selecting a single clone colony to extract a plasmid, performing PCR verification and sequencing identification, and obtaining a transformed bacterium containing a carrier of the target gene. 4. method according to claim 1, is characterized in that: the sequence of the 12 primer part of the target gene forward primer IF described in step a is identical with the sequence of target gene 5 ' end 20 bases; IR I4 is reverse complementary to the sequence of 20 bases at the 3' end of the target gene. 5. The method according to claim 2, characterized in that the bacterium is Escherichia coli. 6. The method according to claim 5, characterized in that the Escherichia coli is DMT competent Escherichia coli. 7. The method according to claim 1, characterized in that the mixing and standing of the step C is carried out at room temperature for 30-90 minutes. 8. The method according to claim 7, characterized in that the mixing time is 30 to 60 minutes. 9. The method according to any one of claims 1-8, characterized in that the target vector is a plasmid.