Specific primer, application and method for efficiently synthesizing and amplifying adenovirus genome DNA

Abstract

The invention relates to the technical field of biology, in particular to a specific primer, application and a method for efficiently synthesizing and amplifying adenovirus genome DNA. The nucleotide sequence of a specific primer used in the method is as shown in SEQ ID NO: 1. The specific primer is designed according to an ITR region of an adenovirus genome, can be efficiently combined with template DNA, can avoid non-specific amplification, and ensures the accuracy of an amplification product; the method is high in amplification efficiency, simple in reaction condition, low in template quality requirement, low in cost, simple to operate, high in efficiency and good in effect, and has wide application prospects in the fields of gene therapy, vaccine development, gene delivery and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to specific primers, applications and methods for efficiently synthesizing and amplifying adenovirus genome DNA. Background Art

[0002] Adenovirus is a non-enveloped double-stranded DNA virus with a genome size of approximately 26-48 kb. Adenovirus genomic DNA has important value in biomedical research and applications, especially in gene therapy, vaccine development, and gene delivery. Adenovirus vectors have become one of the most commonly used viral vectors in gene therapy due to their efficient gene transduction ability, wide host range, and relatively high safety.

[0003] However, efficient synthesis and amplification of adenovirus genomic DNA is a key technical link in realizing the above applications. Whether it is constructing adenovirus vectors, optimizing gene expression, or conducting gene function research, it is necessary to accurately and efficiently synthesize and amplify adenovirus genomic DNA.

[0004] At present, the synthesis and amplification of adenovirus genomic DNA is mainly based on the PAD-easy system, which is an efficient and convenient adenovirus vector construction and amplification technology. However, this method also has some obvious disadvantages in actual operation: the operation steps are cumbersome, and the adenovirus amplification of the PAD-easy system involves multiple steps, such as E. coli amplification, plasmid extraction, linearization and product purification, etc. Each step requires delicate operation and strict quality control, which increases the complexity of the experiment and causes the experimental process to be too long. At the same time, this method requires a large amount of high-purity plasmid extraction, enzymes, product purification and other reagents, which increases the cost of the experiment.

[0005] Polymerase Chain Reaction (PCR) is a core molecular biology technology used to amplify specific DNA fragments. PCR technology simulates the natural replication process of DNA and uses DNA polymerase to quickly amplify target DNA fragments in vitro. Its basic principle includes three steps: denaturation, annealing, and extension, and exponential amplification of DNA fragments is achieved through cyclic reactions.

[0006] PCR technology can amplify target DNA fragments with extremely low template amounts and has extremely high sensitivity. By designing specific primers, the target sequence can be accurately amplified to avoid nonspecific amplification. In addition, PCR technology is also widely used in gene cloning and expression, disease diagnosis, forensic medicine, evolution and taxonomy research, and genome editing.

[0007] Although PCR technology plays an important role in the field of molecular biology, it still faces many technical challenges and limitations when amplifying long DNA fragments (such as adenovirus genomic DNA), mainly including the following aspects:

[0008] (1) Low amplification efficiency: The amplification efficiency of long DNA fragments is significantly lower than that of short DNA fragments. As the length of the amplified fragment increases, the extension ability of the DNA polymerase gradually decreases, resulting in insufficient amplified product. For DNA fragments such as adenovirus genomes that are tens of kb long, conventional PCR technology often has difficulty in achieving efficient amplification.

[0009] (2) High error rate: When amplifying long DNA fragments, DNA polymerase is more likely to introduce mutations, resulting in sequence errors in the amplified product. The adenovirus genome contains highly repetitive sequences and complex secondary structures, which further increases the error rate of PCR amplification and affects the accuracy of subsequent applications.

[0010] (3) Difficulty in primer design: Amplifying long DNA fragments requires the design of efficient and specific primers, which places higher requirements on the length, melting temperature (Tm value) and secondary structure of the primers. The complexity of the ITR (Inverted Terminal Repeat) region and other conserved sequences of the adenovirus genome makes primer design more difficult.

[0011] (4) Complex reaction condition optimization: Long-fragment PCR requires precise optimization of reaction conditions, including enzyme selection, Mg 2+ Concentration, annealing temperature, extension time, etc. The genomes of adenoviruses of different serotypes may differ significantly, further increasing the complexity of optimizing reaction conditions.

[0012] (5) High template quality requirements: Long-fragment PCR has extremely high requirements for the quality and integrity of template DNA. Degradation or contamination of template DNA will significantly affect amplification efficiency. The extraction and purification process of adenovirus genomic DNA is complicated, and it is difficult to ensure a high amount of template.

[0013] (6) Product length limitation: Conventional PCR technology is difficult to amplify DNA fragments larger than 10 kb, while the adenovirus genome is usually 26-48 kb, far beyond the capacity of conventional PCR. Although long-fragment PCR technology (such as LA-PCR) has been developed, its amplification efficiency and accuracy are still not ideal.

[0014] (7) High cost: Amplifying long DNA fragments requires high-fidelity DNA polymerase and special reagents, which increases the experimental cost. For large DNA fragments such as adenovirus genome, multiple attempts to optimize reaction conditions will also significantly increase time and economic costs.

[0015] (8) Complex operation: Long-fragment PCR usually requires multiple steps, including template preparation, primer design, reaction condition optimization, product purification, etc. The experimental process is complicated and has high technical requirements for the operator.

[0016] These shortcomings severely limit its application in adenovirus genomic DNA synthesis and amplification. Although there are some improved methods (such as long fragment PCR, nested PCR, etc.), these methods still have problems such as low efficiency, high error rate, high cost and complex operation. Summary of the invention

[0017] The technical problem to be solved by the present invention is to provide specific primers, their application and a method for efficiently synthesizing and amplifying adenovirus genome DNA.

[0018] The technical solution of the present invention to solve the above technical problems is as follows:

[0019] The present invention provides a specific primer, the nucleotide sequence of the specific primer is shown as SEQ ID NO:1.

[0020] Based on the above technical solution, the present invention can also be improved as follows.

[0021] Furthermore, the Tm value of the specific primer is 60°C.

[0022] The present invention also provides the use of the above-mentioned specific primers in the efficient synthesis and amplification of adenovirus genome DNA.

[0023] The present invention also provides a kit for efficiently synthesizing and amplifying adenovirus genome DNA, comprising the specific primers as described above.

[0024] The present invention provides a method for efficiently synthesizing and amplifying adenovirus genome DNA, which is carried out using the specific primers as mentioned above.

[0025] Further, the following steps are included:

[0026] Obtain adenovirus genomic DNA;

[0027] Using the adenovirus genomic DNA as template DNA and the specific primers to establish a PCR reaction system;

[0028] The PCR reaction system is used to perform synthesis and amplification according to PCR reaction conditions.

[0029] Furthermore, the total volume of the PCR reaction system is 50 μL, including 50 μg of the template DNA and 0.2 μM and 2 μL of the specific primer.

[0030] Furthermore, the PCR reaction system also includes 25ul of 2×PCR Mix, and the total volume of the PCR reaction system is supplemented to 50μL with ddH2O.

[0031] Furthermore, the PCR reaction conditions are: initial denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 56-72°C for 5s, extension at 72°C for 7min, 35 cycles; and final extension at 72°C for 1min.

[0032] Furthermore, the size of the adenovirus genomic DNA is 26-48 kb.

[0033] The beneficial effects of the present invention are:

[0034] (1) The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention, wherein specific primers are designed according to the ITR region of the adenovirus genome, can efficiently bind to the template DNA, and can also avoid nonspecific amplification, thereby ensuring the accuracy of the amplified product;

[0035] (2) The method of the present invention for efficiently synthesizing and amplifying adenovirus genomic DNA has high amplification efficiency and can amplify long fragments of adenovirus genomic DNA;

[0036] (3) The method of the present invention for efficiently synthesizing and amplifying adenovirus genomic DNA has simple reaction conditions, low requirements on template quality, and is applicable to adenovirus genomes of various serotypes;

[0037] (4) The method of the present invention for efficiently synthesizing and amplifying adenovirus genomic DNA has low cost, simple operation, high efficiency and good effect, and has broad application prospects in the fields of gene therapy, vaccine development and gene delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The electrophoresis diagram obtained in Example 1 is a method for efficiently synthesizing and amplifying adenovirus genomic DNA according to the present invention, where M represents a marker, and 1-6 correspond to primers 1-6 in Table 1, respectively;

[0039] Figure 2 The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention, the electrophoresis diagram of different primer concentrations in Example 2;

[0040] Figure 3 The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention, the electrophoresis diagram of different template concentrations in Example 2;

[0041] Figure 4The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention, in Example 3, the electrophoresis diagram of the purified product, M represents a marker, P represents a PCR recovered product, and D represents a PAD-easy system product;

[0042] Figure 5 The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention, the result diagram of first generation sequencing in Example 3;

[0043] Figure 6 The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention, in Example 3, is a graph showing the result of the second generation sequencing;

[0044] Figure 7 The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention, in Example 4, the transfection fluorescence microscope image, Figure 7 A in the middle is the PCR amplification product, Figure 7 B in the middle is the product of the PAD-easy system;

[0045] Figure 8 The test results of Example 5 are as follows: Figure 8 A in the middle is the plaque image produced by the progeny virus. Figure 8 Middle B is a fluorescence microscopy image of newly cultured HEK293 cells. DETAILED DESCRIPTION

[0046] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0047] The nucleotide sequence of the specific primers for efficiently synthesizing and amplifying adenovirus genome DNA of the present invention is shown in SEQ ID NO:1.

[0048] The specific primers of the present invention are designed according to the ITR region of the adenovirus genome, can efficiently bind to the template DNA, and can also avoid nonspecific amplification to ensure the accuracy of the amplified product; the specific primers can efficiently synthesize and amplify large fragments of adenovirus genome DNA.

[0049] Preferably, the Tm value of the specific primer is 60°C.

[0050] The kit for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention comprises the specific primers of the present invention.

[0051] The method for efficiently synthesizing and amplifying adenovirus genomic DNA of the present invention is carried out using the specific primers as described above.

[0052] Preferably, the method of the present invention comprises the following steps:

[0053] S1. Obtain adenovirus genomic DNA; the size of adenovirus genomic DNA is 26-48 kb.

[0054] S2. Using adenovirus genomic DNA as template DNA and the specific primers, a PCR reaction system is established.

[0055] The total volume of the PCR reaction system is 50 μL, including 50 μg of template DNA and 0.2 μM and 2 μL of specific primers; the content of the template DNA and the content of the specific primers can further improve the efficiency of synthesis and amplification while ensuring accuracy and specificity.

[0056] Preferably, the PCR reaction system further includes 25ul of 2×PCR Mix, and the total volume of the PCR reaction system is supplemented to 50 μL with ddH2O.

[0057] S3, using the PCR reaction system to perform synthesis and amplification according to PCR reaction conditions.

[0058] Preferably, the PCR reaction conditions are: initial denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 56-72°C for 5s, extension at 72°C for 7min, 35 cycles; and final extension at 72°C for 1min.

[0059] The synthesis and amplification method of the present invention uses conventional PCR reagents and equipment, and compared with the PAD-easy system commonly used in the prior art, effectively reduces the experimental cost.

[0060] At the same time, the method of the present invention also has the advantages of simple operation and wide applicability, and can be used for the synthesis and amplification of adenovirus genomes of various serotypes.

[0061] The present invention is described below by means of specific examples.

[0062] The template DNA used in the following examples is cmv-gp19k-EGFP adenovirus plasmid, purchased from Wuhan Kaidejinuo Biotechnology Co., Ltd., with a known sequence and a length of about 36k bp. The reagents are a high-fidelity PCR kit (Novizan P510), agarose, a DNA product purification kit, and a transfection reagent (Lipofectamine 3000). The cells are HEK293 cells, and the equipment includes a PCR instrument, an electrophoresis instrument, a gel imaging system, and a cell culture incubator.

[0063] Example 1 Screening of specific primers

[0064] To ensure the specificity and reliability of PCR amplification, multiple primers were screened in this example to obtain primers that can produce a single clear band under specific conditions. Six primers were designed in this example, with Tm values ​​of 40-65°C. The specific sequences are shown in Table 1.

[0065] Table 1

[0066]

[0067] The above primers were used to carry out PCR reaction, and the reaction system and reaction conditions were as follows:

[0068] Reaction system (50 μL): template DNA (50 μg), primers (0.2 μM, 2 μL), 2×PCR Mix (25 ul), and ddH2O to make up to 50 μL.

[0069] Reaction conditions: initial denaturation (98°C (30S)); 35 cycles (denaturation: 98°C, 10S; annealing: 56-72°C, 5S; extension: 72°C, 7min); final extension (72°C, 1min).

[0070] After the PCR reaction is completed, the amplified product is detected by 1% agarose gel electrophoresis. The amplification efficiency and product specificity are analyzed by gel imaging system. The primer ITR-60 that can produce specific bands and the highest efficiency is screened out. The electrophoresis bands are as follows Figure 1 shown.

[0071] The results showed that the primer pair with a Tm value of 60°C produced a single clear band under the above conditions. The band size was consistent with the expected target fragment (about 36k bp), and no nonspecific amplification products or primer dimers were detected by agarose gel electrophoresis.

[0072] Example 2 Optimization of specific primer concentration and template DNA concentration

[0073] This example is based on the specific primer ITR-60 screened in Example 1, and optimizes the template DNA concentration and specific primers in the PCR amplification process.

[0074] Specifically, in this embodiment, the template DNA concentration gradient is set to: 0.1 μg, 1 μg, 10 μg, 20 μg, 50 μg, and 100 μg.

[0075] The concentration gradient of the specific primer ITR-60 set in this embodiment is: 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, and 0.4 μM.

[0076] According to the above concentration gradients, PCR reactions were performed, gels were run and amplification efficiency was detected, and the best template DNA concentration and the best specific primer ITR-60 concentration were selected. The electrophoresis diagrams were as follows: Figure 2 and Figure 3 shown.

[0077] According to the electrophoresis diagram, the best concentration of specific primer ITR-60 is 0.2 μM, and the best concentration of template DNA is 50 ng.

[0078] For the optimal specific primer ITR-60 concentration and the optimal template DNA concentration obtained by the above screening, nucleic acid electrophoresis was performed, and the results were as follows: Figure 4 As shown. Nucleic acid electrophoresis showed that under this condition, the amplified product band was single and bright, proving that the optimized PCR reaction conditions were efficient, stable, and reliable for target gene amplification.

[0079] Example 3 PCR product purification and sequencing verification

[0080] Amplification was performed using the specific primer ITR-60 screened in Example 1 and the optimal specific primer ITR-60 concentration and template DNA concentration screened in Example 2, and the product was purified. To verify the accuracy of the PCR amplification product, the PCR product was sequenced using first-generation (Sanger) sequencing and second-generation (NGS) sequencing technology.

[0081] The specific experimental process is to purify the PCR product using a DNA purification kit (Tiangen, DP205) according to the instructions of the reagent manufacturer, and detect the purity and concentration of the purified product by agarose gel electrophoresis.

[0082] Sequence the purified product, compare the sequencing results with the target sequence, and verify the accuracy and error rate of the amplified product. Figure 5 and 6 shown.

[0083] Sanger sequencing results showed that the sequence of the PCR product was completely consistent with the target gene sequence, and no mutations, insertions or deletions were found. NGS sequencing further confirmed that all reads were highly matched with the target sequence, with a variation frequency of less than 0.01%, indicating that the PCR product sequence was accurate.

[0084] Example 4 Transfection Experiment

[0085] HEK293 cells were cultured at 37°C and 5% CO2.

[0086] The cells were cultured at 2 × 10 5The density of each well was plated in a 6-well plate. The next day, after the cells adhered to the wall, 5 μg of the purified PCR product was mixed with a transfection reagent (such as Lipofectamine 3000) and transfected into a vial of HEK293 cells according to the instructions. At the same time, 5 μg of adenovirus plasmid linearized by PACI produced by PAD-easy was also transfected as above.

[0087] Detection of transfection efficiency: 48 hours after transfection, observe GFP expression by fluorescence microscopy. Extract cell DNA and detect the presence of adenovirus genome by PCR. The fluorescence detection results compared with E. coli production are as follows Figure 7 The results showed that the efficiency of the two transfection methods was comparable.

[0088] Example 5 Progeny virus production verification

[0089] This example verifies whether the genomic DNA amplified by PCR of the present invention can produce progeny viruses after transfection.

[0090] Experimental process of this example I: 7 days after the transfection PCR recovery product, the cell supernatant and cells were collected and frozen and thawed three times. The fluorescence of HEK293 was observed under a fluorescence microscope to prove whether live virus with infectious ability was produced.

[0091] like Figure 8 As shown, obvious cytopathic effect (CPE) was observed in the transfected HEK293 cells. The cell residues were collected and freeze-thawed. Subsequently, the frozen and thawed cell residues were filtered through a 0.22um filter and added to a new HEK293 cell culture system. After 48 hours, GFP fluorescence was observed in the newly cultured HEK293 cells under a fluorescence microscope. This result proves that the progeny virus has been successfully produced.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A specific primer, characterized in that: The nucleotide sequence of the specific primer is shown in SEQ ID NO:

1.

2. A specific primer according to claim 1, characterized in that: The Tm value of the specific primer is 60°C.

3. Use of the specific primers as claimed in claim 1 or 2 in the efficient synthesis and amplification of adenovirus genomic DNA.

4. A kit for efficiently synthesizing and amplifying adenovirus genomic DNA, characterized in that: Comprising the specific primer according to any one of claims 1 or 2.

5. A method for efficiently synthesizing and amplifying adenovirus genomic DNA, characterized in that: The method is carried out using the specific primers as described in any one of claims 1 or 2.

6. A method for efficiently synthesizing and amplifying adenovirus genomic DNA according to claim 5, characterized in that: The following steps are involved: Obtain adenovirus genomic DNA; Using the adenovirus genomic DNA as template DNA and the specific primers to establish a PCR reaction system; The PCR reaction system is used to synthesize and amplify the template DNA according to PCR reaction conditions.

7. A method for efficiently synthesizing and amplifying adenovirus genomic DNA according to claim 6, characterized in that: The total volume of the PCR reaction system is 50 μL, including 50 μg of the template DNA and 0.2 μM and 2 μL of the specific primer.

8. The method for efficiently synthesizing and amplifying adenovirus genomic DNA according to claim 7, characterized in that: The PCR reaction system also includes 25ul of 2×PCR Mix, and ddH2O is used to make up the total volume of the PCR reaction system to 50μL.

9. A method for efficiently synthesizing and amplifying adenovirus genomic DNA according to any one of claims 5 to 8, characterized in that: The PCR reaction conditions are: initial denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 56-72°C for 5s, extension at 72°C for 7min, 35 cycles; and final extension at 72°C for 1min.

10. The method for efficiently synthesizing and amplifying adenovirus genomic DNA according to claim 9, characterized in that: The size of the adenovirus genomic DNA is 26-48 kb.

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