Universal cloning method for short-fragment DNA (deoxyribonucleic acid) based on reverse PCR (polymerase chain reaction) and template-free PCR and application of universal cloning method

Abstract

The invention relates to the technical field of DNA cloning, and discloses a universal short-fragment DNA cloning method based on reverse PCR and template-free PCR and application of the universal short-fragment DNA cloning method, and the universal short-fragment DNA cloning method comprises the following cloning steps: taking an annular plasmid as a template, and adopting reverse PCR amplification to obtain a linearized vector; an upstream primer and a downstream primer are designed and synthesized, the 3'ends of the two primers have complementary overlapping regions, the 5 'ends of the two primers contain plasmid homologous sequences, and the two primers are subjected to thermal cycle self-assembly and extension in a PCR reaction system without exogenous template DNA to form a blunt-end double-stranded DNA fragment; and purifying the blunt-end double-stranded DNA fragment and a linearized vector, connecting through homologous recombination, and transforming a host cell to obtain a recombinant plasmid containing a target gene. The universal cloning method for the short-fragment DNA does not depend on any restriction enzyme cutting site, and the vector is universal; meanwhile, an exogenous template DNA is not needed; time and cost are greatly reduced, and the method is suitable for construction of high-throughput sgRNA, epitope tags, promoter elements and the like.

Description

Technical Field

[0001] This invention relates to the field of gene editing, and in particular to a universal cloning method for short DNA fragments based on inverse PCR and template-free PCR, and its applications. Background Technology

[0002] DNA cloning is a biotechnology that uses in vitro recombination techniques to combine DNA fragments from different sources with a vector to form recombinant molecules, which are then introduced into host cells and amplified to obtain large quantities of identical DNA. In a typical DNA cloning process, the target gene or other DNA fragment is usually inserted into a circular DNA fragment called a plasmid. Insertion is accomplished using restriction endonucleases and DNA ligases, resulting in a single recombinant DNA molecule, or a DNA molecule assembled from fragments from multiple sources.

[0003] Specifically, the circular plasmid and the target gene are cut separately using restriction endonucleases to produce complementary sticky ends. Then, they are ligated using DNA ligase to obtain a recombinant plasmid containing the target fragment. This recombinant plasmid is transformed into competent bacteria, plated on plates containing the appropriate antibiotics to screen positive clones, and then expanded after PCR verification. The host bacteria replicate the plasmid with each division, thus amplifying the target DNA in large quantities. Conventional PCR or OE-PCR requires a template, is lengthy, and is prone to introducing mutations. Commercial gBlock is costly and has a long lead time. Patent CN202110499606.4 discloses a short fragment cloning method that does not rely on PCR, employing a "PCR-free annealing + homologous recombination" strategy. However, when preparing the linearized vector, it requires single or double enzyme digestion to linearize the vector, thus relying on specific restriction sites on the vector and failing to address the problem of low in vivo fragment repair efficiency. Patent CN202510328569.9 requires first digesting the linearized vector with enzymes and designing 15–29bp homologous arms, then synthesizing multiple long primers in segments. The process is cumbersome, time-consuming, and the lower limit of the fragment is limited to 50 bp.

[0004] To address the aforementioned technical problems, this invention provides a three-in-one solution of "reverse PCR linearization vector + template-free PCR fragment self-assembly + blunt end / directional ligation". Summary of the Invention

[0005] The purpose of this invention is to provide a universal cloning method for short DNA fragments based on reverse PCR and template-free PCR, and its applications, in order to solve the above-mentioned technical problems.

[0006] To achieve the above objectives, this invention provides a universal cloning method for short DNA fragments based on reverse PCR and template-free PCR, comprising the following steps: Step 1: Using a circular plasmid as a template, a linearized vector was obtained by reverse PCR amplification; Step 2: Design and synthesize upstream primer F1 and downstream primer R1. The two primers have a 20-30 nt complementary overlap region at the 3' end and contain plasmid homologous sequences at the 5' end. They self-assemble and extend in a PCR reaction system without exogenous template DNA through thermal cycling to form blunt-end double-stranded DNA fragments. Step 3: After ligating the blunt-end double-stranded DNA fragment to the linearized vector, transform the host cell to obtain a recombinant plasmid containing the target gene.

[0007] Furthermore, the circular plasmids in step 1 include pUC19, pET28a, or pET32a, etc.

[0008] Furthermore, in step 1, reverse PCR amplification uses high-fidelity DNA polymerase, and the amplification product is gel-cleaved and recovered to remove the methylated template.

[0009] Furthermore, the reverse PCR amplification primers for pUC19 are shown in SEQ ID NO.1-2; the reverse PCR amplification primers for pET28a are shown in SEQ ID NO.3-4; and the reverse PCR amplification primers for pET32a are shown in SEQ ID NO.5-6.

[0010] Furthermore, in step 2, the length of the blunt-ended double-stranded DNA fragment is 30-105 bp.

[0011] Furthermore, in step 3, the blunt-end double-stranded DNA fragment is ligated to the linearized vector via in vitro recombination ligation based on homologous sequences.

[0012] Furthermore, in step 3, the molar ratio of blunt-ended double-stranded DNA fragments to linearized vectors is 3:1.

[0013] Furthermore, the present invention also provides the application of the above-mentioned universal cloning method for short DNA fragments based on inverse PCR and template-free PCR in the high-throughput construction of sgRNA boxes, epitope tags or regulatory elements.

[0014] The advantages and positive effects of the universal short DNA cloning method based on reverse PCR and template-free PCR described in this invention and its application are as follows: 1. The short DNA cloning method described in this invention does not depend on any restriction enzyme sites, and the vector is universal; 2. The short-fragment DNA cloning method described in this invention does not require exogenous template DNA, and features zero waiting time, zero contamination, and zero cost; 3. The short-fragment DNA cloning method described in this invention has a positive rate of ≥80% and a sequencing accuracy of ≥90%; 4. The short-fragment DNA cloning method described in this invention has a total experimental cycle of ≤3 working days (1 day for primer synthesis, 1 day for ligation and transformation, and 1 day for sequencing), which significantly shortens the cloning delivery time. 5. The short DNA cloning method described in this invention costs approximately 30-50% of commercially synthesized gene fragments, significantly reducing costs and making it suitable for high-throughput construction of sgRNA, epitope tags, promoter elements, etc.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 This is a flowchart of the technical process in an embodiment of the present invention (reverse PCR → template-free PCR → ligation → transformation). Figure 2 These are electrophoresis images of the linearized reverse PCR of different vectors (pUC19, pET28a, pET32a) in the embodiments of the present invention; Figure 3 The above are the electrophoresis results of template-free PCR products of the target fragment in the embodiments of the present invention, wherein A is the electrophoresis result of template-free PCR product of fragment PUC19 plasmid, B is the electrophoresis result of template-free PCR product of fragment pET28a plasmid, and C is the electrophoresis result of template-free PCR product of fragment pET32a plasmid. Figure 4 Sequencing results of the PUC19-30bp recombinant plasmid in this embodiment of the invention; Figure 5 Sequencing results of the PUC19-50bp recombinant plasmid in this embodiment of the invention; Figure 6 Sequencing results of the PUC19-105bp recombinant plasmid in this embodiment of the invention; Figure 7 Sequencing results of pET28a-30bp recombinant plasmid in the embodiments of this invention; Figure 8 Sequencing results of pET28a-50bp recombinant plasmid in the embodiments of this invention; Figure 9 Sequencing results of pET28a-105bp recombinant plasmid in the embodiments of this invention; Figure 10 Sequencing results of pET32a-30bp recombinant plasmid in the embodiments of this invention; Figure 11 Sequencing results of pET32a-50bp recombinant plasmid in this embodiment of the invention; Figure 12 Sequencing results of pET32a-105bp recombinant plasmid in this embodiment of the invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0019] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0020] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0021] The pUC19, pET28a, and pET32a vectors are plasmids preserved by the Veterinary Pharmacology Group of Nanjing Agricultural University, and can be obtained from the public upon request.

[0022] This invention discloses a universal method for cloning short DNA fragments based on inverse PCR and template-free PCR, comprising the following steps (flowchart shown below). Figure 1 (as shown) Step 1, Preparation of linearized vector by reverse PCR: a) Using any circular plasmid as a template, design a pair of reverse primers (VF / VR), 20-25nt, Tm≈60℃, to amplify the entire vector; b) Reaction system: 50 ng plasmid, high-fidelity PCR Mix, upstream primer, downstream primer, add water to 50 µL; c) Program: 95℃ for 3 min; 35 cycles (95℃, 15s→60℃, 15s→72℃, 1min / kb (based on high-fidelity enzyme)); 72℃ for 5 min; d) Agarose gel electrophoresis → gel cutting and purification → obtaining linearized vector (with blunt ends or retaining custom ends).

[0023] Step 2, template-free PCR self-assembly of short fragments: a) Design upstream primer F1 and downstream primer R1, both of which contain a 15-30 nt complementary overlap region at their 3' ends and a plasmid homologous sequence (15-30 nt) at their 5' ends. b) Reaction system: 1µM each of F1 and R1, 25µL of high-fidelity PCR Mix, and water added to 50µL; c) Program: 95℃, 3 min; 35 cycles (95℃, 15s→60℃, 15s→72℃, 10s); 72℃, 5 min; d) Results: After annealing, the two primers extended to form blunt-ended double-stranded DNA fragments; e) Agarose gel electrophoresis → gel cutting and purification → obtaining the target fragment.

[0024] Step 3, Connection and Transformation: a) Take 50 ng of the target fragment + 25 ng of the linearized vector (molar ratio ≈ 3:1), add homologous recombinase, and incubate at 60℃ for 5 min; b) 10 µL of ligation solution was used to convert 100 µL of DH5α, which was then spread onto LB plates containing the corresponding antibiotic and incubated overnight at 37°C. c) Colony PCR identification; positive clones are sent for sequencing.

[0025] Example To verify the feasibility, universality, and accuracy of the short DNA cloning method based on the combination of reverse PCR and template-free PCR provided in this invention, detailed explanations are given using pUC19, pET28a, and pET32a vectors as examples. Table 1 shows the target fragment and the primer sequences used.

[0026] Table 1. Target fragment and primer sequences (5'→3')

[0027] 1. Selection of experimental materials and carriers: Three commonly used plasmid vectors were selected as the cloning backbone: pUC19 (high copy number, commonly used for routine cloning and blue-white screening). pET28a (T7 promoter, commonly used for protein expression); pET32a (a fusion tag expression system, including Trx tags); All of the above vectors are circular double-stranded DNA, without any restriction endonuclease treatment, and are used directly for inverse PCR linearization.

[0028] 2. Reverse PCR linearization vector: Using the reverse primers listed in Table 1 (reverse PUC19-F, reverse PUC19-R, reverse pET28a-F, reverse pET28a-R, and reverse pET32a-F, reverse pET32a-R) as amplification primers, reverse PCR amplification was performed on the three plasmids respectively. The reaction systems are as follows (Table 2): Table 2 Reverse PCR reaction system

[0029] The PCR reaction procedure is as follows: 95℃, 3 min; 35 cycles (95℃, 15 s→58℃, 15 s→72℃, 1 min / kb (based on high-fidelity enzyme)); 72℃, 5 min.

[0030] The amplified product was detected by 1% agarose gel electrophoresis, and a clear single band was visible, consistent with the expected size. Figure 2 As shown in the figure, the linearized vector was obtained after gel purification and used for subsequent ligation.

[0031] 3. Template-free PCR synthesis of the target fragment: Design and synthesize upstream and downstream primers for 30 bp, 50 bp, and 105 bp target fragments (sequences are shown in Table 1). The 3' end of the primers contains a 15-30 nt complementary region, and the 5' end contains a 15-30 nt sequence homologous to the target vector.

[0032] The template-free PCR reaction system is as follows (Table 3): Table 3

[0033] The PCR reaction procedure is as follows: 95℃, 3 min; 35 cycles (95℃, 15s→60℃, 15s→72℃, 10s); 72℃, 5 min.

[0034] The reaction products were detected by 3% agarose gel electrophoresis, and clear bands were visible. Figure 3 As shown in the image, this indicates that the two primers successfully self-assembled and extended into a double-stranded DNA fragment. The target fragment was obtained after gel purification and used for subsequent ligation.

[0035] 4. Homologous recombination and transformation: The purified target fragment was mixed with the linearized vector at a molar ratio of 3:1 (approximately 50 ng fragment + 25 ng vector), and a commercial homologous recombinase (such as Ezmax® Ultra Universal CloneMix) was added. The mixture was then reacted at 60°C for 5 min.

[0036] Transform 10 μL of the ligation product into 100 μL of DH5α competent cells, plate them on LB agar plates containing the appropriate antibiotics, and incubate overnight at 37°C.

[0037] 5. Screening and sequencing validation of positive clones: Six to ten single colonies were randomly selected from each plate for colony PCR identification. Universal sequencing primers were used (PUC19-F, PUC19-R, T7-F, and T7-R are shown in Table 1). The PCR products were detected by agarose gel electrophoresis, and the positive rate was ≥90%.

[0038] The positive clones were then sent for sequencing, and the sequencing results were completely consistent with the expected sequence. Figures 4-12 As shown in the figure, there are no mutations or insertions / deletions, indicating that the method has high fidelity and correctness.

[0039] The method described in this invention has the following advantages: 1) The cloning process does not depend on any restriction enzyme sites, and the vector is universal; 2) No exogenous template DNA required, zero waiting time, zero contamination, and zero cost; 3) Positive rate ≥ 80%, sequencing accuracy ≥ 90%; 4) The entire experimental cycle is ≤3 working days (1 day for primer synthesis, 1 day for ligation and transformation, and 1 day for sequencing), which significantly shortens the clone delivery time.

[0040] 5) The cost is about 30-50% of that of commercially synthesized gene fragments, which greatly reduces the cost and makes it suitable for high-throughput construction of sgRNA, epitope tags, promoter elements, etc.

[0041] Therefore, the universal short DNA cloning method based on reverse PCR and template-free PCR described in this invention does not rely on any restriction enzyme sites and does not require exogenous template DNA; it is short in time, low in cost, and suitable for high-throughput construction of sgRNA, epitope tags, promoter elements, etc.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A universal cloning method for short fragments of DNA based on inverse PCR and template-free PCR, characterized in that, The method comprises the following steps: Step 1: linearized vector is obtained by reverse PCR amplification using a circular plasmid as a template; Step 2: an upstream primer and a downstream primer are designed and synthesized, the two primers have a 20-30 nt complementary overlap region at the 3' end, and the 5' end contains a plasmid homologous sequence, and a blunt-end double-stranded DNA fragment is formed by self-assembly and extension in a PCR reaction system without an external template DNA through thermal cycling; Step 3: the blunt-end double-stranded DNA fragment is connected to the linearized vector through homologous recombination after purification, and a host cell is transformed to obtain a recombinant plasmid containing a target gene.

2. The universal cloning method for short DNA fragments based on inverse PCR and template-free PCR according to claim 1, wherein: The circular plasmid in step 1 includes pUC19, pET28a or pET32a.

3. The universal cloning method for short DNA fragments based on inverse PCR and template-free PCR according to claim 2, characterized in that: The reverse PCR amplification primers of pUC19 are shown as SEQ ID NO. 1-2; the reverse PCR amplification primers of pET28a are shown as SEQ ID NO. 3-4; and the reverse PCR amplification primers of pET32a are shown as SEQ ID NO. 5-6.

4. The universal cloning method for short DNA fragments based on inverse PCR and template-free PCR according to claim 1, wherein: In step 2, the length of the blunt-end double-stranded DNA fragment is 30-105 bp.

5. The universal cloning method for short DNA fragments based on inverse PCR and template-free PCR according to claim 1, characterized in that: In step 3, the connection mode of the blunt-end double-stranded DNA fragment and the linearized vector is in-vitro recombination connection based on homologous sequences.

6. The universal cloning method for short DNA fragments based on inverse PCR and template-free PCR according to claim 1, wherein: In step 3, the molar ratio of the blunt-end double-stranded DNA fragment to the linearized vector is 3:

1.

7. Application of the short fragment DNA universal cloning method based on reverse PCR and template-free PCR in any one of claims 1-6 in high-throughput construction of sgRNA cassettes, epitope tags or regulatory elements.

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