CN106047912B

CN106047912B - Novel gene cloning method

Info

Publication number: CN106047912B
Application number: CN201610357602.1A
Authority: CN
Inventors: 王超
Original assignee: Beijing Clone Ouke Technology Co Ltd
Current assignee: Beijing Clone Ouke Technology Co Ltd
Priority date: 2016-05-26
Filing date: 2016-05-26
Publication date: 2020-01-10
Anticipated expiration: 2036-05-26
Also published as: CN106047912A

Abstract

The invention relates to a gene cloning technology, which is characterized in that a carrier basic element and a target gene are assembled at one time by introducing a fixed joint in a gene cloning process, universal pcr primers are designed by using the fixed joints as molecular markers for screening and sequencing, and simultaneously, antibodies specific to the universal fixed joints are used for carrying out target gene expression detection. The fixed joint is a DNA joint (simultaneously, a DNA joint for clone connection) with a universal sequence fixed in different clone experiments, and then the oligopeptide coded by the fixed clone joint is used as a universal detection marker.

Description

Novel gene cloning method

One, the technical field

The invention belongs to the field of genetic engineering, and particularly relates to a gene cloning method.

Second, background Art

Gene cloning techniques have been known for nearly 40 years since 1967-. The most traditional and classical gene cloning technology is to treat the vector and the target gene with restriction enzyme separately, and transform the vector and the target gene into competent Escherichia coli cells after ligation with ligase. As one of the most fundamental technologies of molecular biology and biomedicine, the gene cloning technology has a huge market potential all the time, and a plurality of well-known enterprises and large biopharmaceutical companies, such as NEB, Roche, Thermo scientific and the like, carry out deep and extensive research on the technology. At present, certain development is provided in the aspects of cloning vector types and finding and applying of endonuclease.

In the aspect of cloning vectors, the research progress is mainly embodied in that the number of the developed vectors is more and more, and the types of the developed vectors are more and more abundant. There are 395 vectors currently available to users from Life Technology, covering various types of vectors that can be expressed in bacterial, fungal, insect and mammalian cells; likewise, Novagen also developed a number of new cloning vector products, and 162 are currently available for selection by the user. In general, these vectors contain new molecular signatures, specific resistance genes, and different transcription promoters to satisfy different experimental requirements of different users. Wherein 33 different molecular tags are included in the vector provided by Life Technology; 11 different resistance genes, and 32 different transcription promoters. Although the number and the variety of the carriers which can be selected by experimenters are more and more, and the variety is more and more abundant, few technologies are available at present, and users can independently select and assemble the carrier framework which meets the experimental requirements of the users.

In the aspect of restriction endonucleases, the research progress is mainly reflected in that the developed endonucleases are more and more in quantity and more in variety. The total number of endonuclease that can be provided by NEB company at present is 708, of which 302 restriction endonucleases are available for gene cloning. Likewise, Thermo Scientific can provide at least 191 restriction enzymes for the user to select for gene cloning. In addition to research on new endonucleases, more and more research is also focused on developing new enzyme digestion buffers to reduce the activity of asterisks and increase the speed of enzyme reactions. NEB introduced a newer version of the enzyme reaction RE-Mix in 2012^TM。RE-Mix^TMThe conventional enzyme digestion components are prepared into a premixed solution containing a special catalyst, so that the reaction speed is accelerated, DNA and water are simply added during the enzyme digestion reaction without adding other components, and the operation steps are simplified. Meanwhile, the improvement also avoids errors and calculation errors of manual premixing, reduces pollution caused by manual operation, and effectively solves the problem of time of enzyme digestion reaction. But the enzyme digestion reaction is accelerated, the specificity of the enzyme digestion reaction is also reduced, and the success rate of gene cloning is directly influenced.

Although the development of the traditional gene cloning technology is promoted to a certain extent by the research progress of cloning vectors and restriction endonucleases, the technical principle still depends on two indispensable operations of restriction endonuclease digestion of target gene DNA fragments and plasmid vectors, and ligase ligation of target genes and plasmids. Therefore, the success of gene cloning depends on the efficiency of the restriction endonuclease digestion reaction and the DNA ligase reaction. Although the percentage of positive clones could be increased by removing the 5' phosphate group from the linearized vector with alkaline phosphomonoesterase, the procedure was complicated, the technique was difficult to master, and often failed to function. To overcome the drawbacks of conventional gene cloning methods, companies have begun to develop ligase-independent cloning methods, typically represented by recombinase-based "In-fusion" cloning techniques, or seamless ligation cloning techniques.

The In-fusion cloning technology is a new cloning technology recently introduced by some biotechnology companies. The technical principle is that a plurality of base sequences corresponding to the tail end of a linearized vector are introduced during the design of a PCR primer, and after DNA recombinase is added, a target gene segment and the vector are fused at a homologous sequence (usually more than 25 bases), so that the tail end of the target gene and the tail end of the vector are replaced, and the purpose of gene cloning is achieved. Through recombinase replacement, the In-fusion technology avoids restriction enzyme and ligase In the traditional gene cloning, simplifies the experimental operation and improves the experimental efficiency. However, the technology still needs restriction endonuclease to carry out linearization treatment on the target vector, and tapping and purification are needed at the same time, so the operation is still complicated; in addition, when the technology needs to combine the sequence of the carrier to design the primers at the two ends of the PCR, bases at the tail end of the linearized carrier are respectively added at the two sides of the homologous sequence of the target gene, and the homologous base sequences added in different experiments are different, so the PCR primer design program is complex and is not beneficial to the automation and programmed management by a computer. Because the homologous base sequences added in different experiments are different, different PCR primers are also needed for screening positive clones of different gene cloning experiments in the gene cloning process, and corresponding different but specific antibodies are also needed in the process of detecting different target gene expression products.

Description of the drawings

FIG. 1: the gene cloning technology of the invention is a schematic diagram. Different cloning empty vectors are subjected to functional modularization by adding fixed universal DNA sequences (namely fixed joints) at the tail ends of universal elements (promoters, terminators and drug-resistant genes) of different vectors, and then vectors containing target genes, which can meet the requirements of different experimental purposes, are assembled at one time by using fewer raw materials through selection and combination of functional modules of the vectors.

FIG. 2A: different genes are loaded into the same vector; FIG. 2B the same gene assembled into different vectors. Because each carrier basic element and the target gene segment contain a pre-designed matched fixed DNA joint, different target genes can be assembled into the same carrier in a large quantity in the same batch; meanwhile, PCR products of the same target gene can be assembled into other types of vectors according to experimental requirements.

FIG. 3A: the gene cloning operation principle diagram of the invention; FIG. 3B: examples of the design of the gene cloning adaptor of the present invention are shown.

FIG. 4A: cloning the fixed joint cassette A into a vector pEX18Gm through the traditional HindIII enzyme digestion connection to obtain a clone body; FIG. 4B: PCR primers specific for X1234 containing an immobilized linker (red label) were used for PCR amplification; FIG. 4C: carrying out PCR positive clone screening by using a universal PCR primer containing a fixed joint; FIG. 4D: DNA sequencing results of 6 single clones.

FIG. 5A: taking the gene PA0022 of the clone pseudomonas aeruginosa as an example, two gene segments are cloned at one time; FIG. 5B: PCR primers specific to PA0022 containing a fixed linker (red and blue labels) were used for PCR amplification, then directly transformed into E.coli and screened with drugs;

FIG. 5C: carrying out PCR screening on 14 randomly selected monoclonals by using a PCR primer specific to the fixed joint;

FIG. 5D: DNA sequencing results of 14 clones.

FIG. 6 is a schematic diagram of the functional modularization (i.e. introduction of a fixed joint by enzyme digestion or homologous recombination) of the basic elements (promoter, terminator and drug-resistant gene) of the cloning vector and the reassembly of the basic elements and the target gene containing the corresponding fixed joint into a new plasmid at a time.

FIG. 7A-FIG. 7D an example of the assembly and successful use of the basic elements of the cloning vector:

FIG. 7A: schematic diagram of preparation assembly example of cloning vector basic elements;

FIG. 7B: examples of primer sequences;

FIG. 7C: carrying out PCR positive clone screening by using a universal PCR primer containing a fixed joint;

FIG. 7D: and (5) DNA sequence identification.

Fourth, the invention

The inventor of the present application found in the development process that the fixed joints can be introduced in the gene cloning process to assemble the basic elements of the vector and the target gene at one time, and the fixed joints are used as molecular markers to design universal pcr primers for screening and sequencing gene cloning, and simultaneously, the antibodies specific to the universal fixed joints or the amino acid sequences coded by the fixed joints are used for detecting the expression of the target gene. The fixed joint in the invention can be defined as a DNA sequence which can be universally used in different cloning experiments and can be fixed by sequences, and is the DNA joint of each carrier basic element and two ends of a target gene when gene cloning is carried out. In the gene cloning process, the DNA sequence of the fixed joint can be used for designing a universal PCR primer, and the coded amino acid sequence can also be used as a universal detection marker for target gene expression. Therefore, all the target genes cloned by the technology of the invention can be detected by using the antibody of the universal molecular tag. Meanwhile, the fixed joint can also be used as a universal PCR primer during clone screening, so that the problem that different PCR primers are needed for positive clone screening of different gene clone experiments in the traditional gene cloning process is solved. It is to be noted that the length of the immobilization linker may be between 10 and 35 nucleotides in the present invention, but is preferably between 12 and 18 nucleotides. In designing the fixed linker, the nucleotide can be selected to be composed according to experimental requirements, and once the design is completed, it may not need to be changed in subsequent experiments. This assembly can be achieved by enzymatic cleavage to generate base complementary ends as described in the embodiments of the present invention, or by homologous recombination by a recombinase. One condition to be met when designing these fixed joints is that these fixed joints can be used for assembly of the carrier basic elements. The basic elements of the vector in the present invention can be defined as DNA fragments containing different functional units of the backbone of the gene cloning vector, or related combinations of these different DNA fragments. These functional fragments may be the origin of replication necessary for the replication of the vector in the cell, the drug-resistant gene in the vector backbone for gene cloning screening, or the promoter and terminator for transcription termination in the vector backbone for activating the gene of interest. A gene of interest is defined in the present invention as one or several DNA fragments that the experimenter wishes to ligate into the vector backbone. While the fixed joints among different types of vector basic elements (such as promoters, drug-resistant genes and terminators) are different among DNA sequences, but the DNA sequences of the fixed joints among the vector basic elements of the same type (compared with the DNA sequences of the fixed joints among the same type of vector basic elements, such as tetracycline-resistant genes, gentamicin-resistant genes and ampicillin genes), so that experimenters can freely select different vector basic elements and target genes to assemble according to the specific experimental requirements, and complete different gene cloning experiments simply, conveniently and in batches at one time (see figure 1).

The technical scheme of the application is as follows: an additional fixed joint of about 15 nucleotides is added during the synthesis of PCR primers, then the basic element of the vector and the target gene are amplified by using the primers with the fixed joints, and the basic element module of the vector with the fixed joints and the target gene fragment are obtained after the PCR product is purified. Besides, these modules of basic elements of vectors with fixed linkers can also be prepared by inserting a short piece of DNA sequence into the empty skeleton of the vector by conventional molecular biology methods, and then cutting by enzyme treatment to obtain fragments of basic elements of vectors with fixed linkers and target gene fragments (see FIG. 1). Based on the reasonable design of these fixed linkers, these fixed linkers can generate specific single-stranded sticky ends under enzyme digestion. By the base complementary principle, these DNA fragments can pair themselves, and can be connected together rapidly, directionally and specifically according to the pre-designed order to form linear or circular DNA molecules. By the method, the function modularization of the traditional DNA carrier and the assembly of the target gene and the DNA carrier element are realized. Under the condition of no need of ligase, the assembled carrier containing the target gene can be transferred into escherichia coli by a chemical conversion method for drug screening and PCR screening, so that the required gene clone can be obtained quickly and efficiently.

By the method, the vectors containing the target DNA fragments, which can meet different target requirements, can be assembled at one time only by selecting and combining different vector general elements (mainly comprising the DNA fragments of a promoter, a resistance gene and a terminator of the vector); the method solves the problems that different empty vectors are needed in different gene cloning experiments in the prior art, so that the empty vectors needed by the experiments have the characteristics of numerous quantities and high cost.

The method of the present application differs from conventional gene cloning methods in that the cloned gene product of interest is typically detected by cloning with additional molecular markers such as FLAG, MYC and V5 followed by hybridization detection using a western blot with the corresponding antibodies. The method of the application has the advantages that the detection marker is universal, so different types of experiments can be screened by using the same PCR primer, and meanwhile, the expression of target genes of different cloning experiments can be detected by using the same antibody. Therefore, the method of the invention simplifies the detection and screening process and can also be operated in batch. In addition, the invention fragments the empty vector skeleton in advance by functional modules, thereby effectively solving the problems of vector self-ligation and empty vector false positive in the traditional cloning. The basic elements of the vectors are selected and freely assembled according to the specific requirements of the experiment, the defect that different experiments in the traditional gene cloning method need different vector frameworks and different vector frameworks need different pre-enzyme digestion is overcome to the maximum extent, and the accuracy and the success rate of the gene cloning experiment are greatly improved.

In the gene cloning method, PCR primers of different target genes have the same fixed joint, and the joint is not associated with a specific DNA sequence of the target gene, so the design difficulty is simplified in the design of the primers; in the aspects of clone screening and target gene expression detection, universal PCR primers and universal specific antibodies can be used, so that the experimental steps are simplified, and batch operation can be realized, thereby having the following advantages:

through the selection and combination of common elements of different carriers, carriers which can meet different purposes are assembled by less raw materials, so that the cost of the experiment is saved (see figure 1);

the primer design for amplifying the target gene is simpler by utilizing the fixed cloning joint, because the variation of a DNA sequence and whether the inside of the target gene contains a proper restriction enzyme cutting site do not need to be considered;

based on the used fixed cloning joint, PCR primers used in cloning screening are designed, and positive clones can be screened in cloning universally;

the oligopeptide coded by the used fixed cloning joint is used as an antigen to produce a specific antibody, and the antibody can be used for universally detecting the expression condition of a target gene;

because each carrier basic element and target gene segment contain a pre-designed fixed DNA joint, different target genes can be assembled into the same carrier in a large quantity in the same batch; meanwhile, PCR products of the same target gene can be assembled into other types of vectors according to experimental requirements without other additional experimental operations (such as the attached figure 2).

Detailed Description

Examples 1,

Step 1, designing the fixed joint of the gene clone according to the steps described in FIG. 3B. The fixed cloning adaptor was designed to contain 3 parts, the Nt. BbvCI cleavage site (green), the SmaI cleavage site (blue) and the variable sequence in between (red). Wherein N represents any one nucleotide of A, G, C, T. The fixed linker of the PCR primers of the gene of interest, which matches the sequence of the fixed linker at the end of the vector, also correspondingly comprises 3 basic parts GG (green) at the 5 'end, CCCTCAGC at the 5' end and variable sequences in between.

Step 2, the gene cloning operation of the present invention is performed according to the steps described in FIG. 3A. And (3) mixing an empty cloning vector skeleton containing the fixed cloning joint in the step (1) with a target gene product, treating for half an hour by using Nt.

Example 2, a specific carrier fixed joint and a fixed joint of a PCR primer of a target gene are designed according to the method in example 1, the fixed joints are used for PCR amplification, and then Escherichia coli is transformed for PCR screening.

A) As shown in fig. 4A: in the embodiment, the case A is respectively determined as CATAG and TGGGA (red); the vector fixed joint cassette A is cloned into a vector pEX18Gm through the traditional HindIII enzyme digestion connection to obtain a cloning vector. This cloning vector was then linearized with SmaI to give the basic elements of the vector that were used (in this example, promoter, terminator and resistance gene were not separated);

B) as shown in fig. 4B: the gene X1234 of the clone of red yeast is exemplified. PCR primers containing a fixed joint and specific to X1234 are used for PCR amplification, and then escherichia coli is directly transformed and screened by using a medicament;

C) as shown in fig. 4C: PCR screening is carried out on 6 randomly selected monoclonals by using a PCR primer specific to a fixed joint, and the result shows that all 6 clones are positive clones;

D) as shown in fig. 4D: the DNA sequencing results showed that all 6 clones were positive clones.

A) As shown in FIG. 5A, two gene fragments were simultaneously cloned at a time, taking PA0022, which is a gene for cloning Pseudomonas aeruginosa, as an example.

B) As shown in FIG. 5B, PCR primers specific to PA0022 containing an immobilized linker were used for PCR amplification, followed by direct transformation of E.coli and drug screening.

C) As shown in FIG. 5C, the randomly selected 14 single clones were then PCR screened with PCR primer pairs specific for the fixed linkers, and all 14 clones were positive clones, in this example, 5-GCTGAGGCATAGCCCTCAGC-3 and 5-GCTGAGGCACCCTGGAGTCGT-3 with universal PCR primer pairs containing fixed linkers.

D) As shown in fig. 5D, DNA sequencing results showed that all 14 clones were positive clones.

Example 4 basic elements (promoter, terminator and drug resistance gene) of the empty cloning vector were functionally modularized and reassembled with the gene of interest into a new plasmid.

As shown in FIG. 6, the cloning vector immobilization sequence set forth in FIG. 6 is one of many possible sets of immobilization sequences. In this example, the gene cloning technology of the present invention automatically assembles 4 different DNA fragments (including vector promoter, terminator, drug resistance gene and target gene) into a new vector according to the pre-designed sequence and direction (as shown in fig. 7), and the results of PCR screening and DNA sequencing show that the cloning efficiency and accuracy of this method is > 99%.

Example 5 in this example, the gene cloning technology of the present invention autonomously assembles more than 4 different DNA fragments (including vector promoter, terminator, drug resistance gene and multiple target genes) into a new vector according to the pre-designed sequence and direction.

As shown in fig. 7C and 7D, PCR screening and DNA sequencing results indicate that the cloning efficiency and accuracy of this method is > 99%.

Claims

1. A method for gene cloning in molecular biology, the method comprising the steps of: designing PCR primers specific to the target gene and basic elements of the vector with a fixed linker of 10-35 nucleotides in length, optionally selected from nucleotides A, G, C and T, and including specific nicking enzyme cutting sites, wherein the DNA sequence of the fixed linker does not need to be changed in subsequent experiments of different batches once the design is completed; (ii) constructing, combining, selecting and synthesizing the cloning vector required by the basic elements of the vector according to the fixed joint; (iii) the fixed linker participates in the whole process of gene cloning and performs protein expression together with the target gene, (iv) cloning PCR screening is performed by using corresponding PCR primers specific to the fixed linker according to the fixed linker; (v) the enzyme antigen produces a specific antibody based on the oligopeptide encoded by the immobilized linker, which is commonly used to detect the expression of all the genes of interest cloned by this method.

2. The method of claim 1, wherein the length of the immobilization linker in (i) is between 12 and 18 nucleotides.

3. The method of claim 1, wherein the vector essential elements in (ii) are constructed by PCR amplification using PCR primers containing an immobilized linker, or by other methods in which an immobilized linker is introduced into the vector backbone followed by enzymatic purification.

4. The method of claim 1, wherein the combination of vector essential elements is performed based on the DNA sequence in the fixed linker, assembly is achieved by nickase cleavage to generate base complementary ends, or homologous recombination is achieved by a recombinase.

5. The method of claim 1, wherein the positive clone screening is performed by using a fixed linker DNA sequence introduced by cloning and designing a corresponding PCR primer.

6. The method as claimed in claim 1, wherein the expression of the target gene is detected by using the corresponding specific antibody derived from the antigen labeled by the molecular marker encoded by the cloned and introduced DNA sequence of the fixed linker.