DNA amplification method, device and system and mRNA preparation method

Abstract

The invention relates to the technical field of DNA amplification, and discloses a DNA amplification method, device and system and a preparation method of mRNA, the method comprises the following steps: carrying out N1 times of PCR circulation in a first PCR reaction solution to obtain a first-stage solution; contacting the first-stage solution with a target solid-phase carrier, so that the target solid-phase carrier is combined with the target DNA product to form a carrier conjugate; separating the carrier conjugate from the first stage solution; eluting the carrier conjugate in a second PCR reaction solution to release the target DNA product from the carrier conjugate, and separating the target solid-phase carrier after the target DNA product is released from the second PCR reaction solution to obtain a second-stage solution; and carrying out N2 times of PCR circulation in the second-stage solution to obtain an amplification solution containing a target DNA product. According to the method, PCR is divided into two stages, impurities and inhibitors are further removed through selective combination of solid-phase carriers and DNA polymerase and mismatch recognition enzyme, and high-fidelity and high-purity target DNA output is achieved.

Description

Technical Field

[0001] This invention relates to the field of DNA amplification technology, and in particular to a DNA amplification method, apparatus, system, and method for preparing mRNA. Background Technology

[0002] Polymerase chain reaction (PCR) is one of the most fundamental and important techniques in molecular biology. PCR typically involves repeated thermal cycling of the reaction mixture between two or three temperatures, achieving milligram-level DNA yields on a large scale. In practice, while PCR cycling offers advantages in purity and accuracy, it faces potential challenges such as host-associated contamination, methylation modifications, and sequence shifts. Specifically, limitations in the accumulation of inhibitors and mismatch extension within the reaction system make it difficult to simultaneously achieve high fidelity, high purity, and microgram-to-milligram-level output within conventional volumes. Therefore, achieving high-fidelity, high-purity target DNA production during PCR cycling remains a key technical challenge in this field. Summary of the Invention

[0003] This invention provides a DNA amplification method, apparatus, system, and mRNA preparation method for achieving high-fidelity, high-purity target DNA production.

[0004] The first aspect of this invention discloses a DNA amplification method, the method comprising: S1. Perform N1 PCR cycles in a first PCR reaction solution containing at least template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme to obtain the first stage solution; S2. Contact the first-stage solution with the target solid-phase support to bind the target solid-phase support with the target DNA product to form a carrier conjugate; separate the carrier conjugate from the first-stage solution; S3. The vector conjugate is eluted in a second PCR reaction solution containing at least primers, dNTPs, DNA polymerase and mismatch recognition enzyme to release the target DNA product. The target solid-phase vector after releasing the target DNA product is separated from the second PCR reaction solution to obtain a second-stage solution. S4. Perform N2 PCR cycles in the second stage solution to obtain an amplification solution containing the target DNA product; Wherein, N1 is a preset first cycle number, and N2 is a preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, CEL I nuclease, EndoMS enzyme, and Surveyor enzyme.

[0005] A second aspect of the present invention discloses a DNA amplification device, the device comprising: A first reaction chamber, comprising at least a first PCR reaction solution containing template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme; the first reaction chamber is used to control the first PCR reaction solution to undergo N1 PCR cycles to obtain a first-stage solution. A purification device is connected to the first reaction chamber. The first-stage solution is brought into contact with the target solid-phase carrier in the purification device to bind the target solid-phase carrier with the target DNA product to form a carrier conjugate. The purification device is used to separate the carrier conjugate from the first-stage solution. The second reaction chamber contains at least a second PCR reaction solution comprising primers, dNTPs, DNA polymerase, and mismatch recognition enzyme. The second reaction chamber is used to elute the vector conjugate in the second PCR reaction solution to release the target DNA product. The second reaction chamber is also used to separate the target solid-phase vector after the release of the target DNA product from the second PCR reaction solution to obtain a second-stage solution. The second-stage solution undergoes N2 PCR cycles in the second reaction chamber to obtain an amplification solution containing the target DNA product; Wherein, N1 is a preset first cycle number, and N2 is a preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, and CEL I nuclease.

[0006] The third invention discloses a DNA amplification system, the system comprising: A liquid processing workstation is used to prepare a first PCR reaction solution containing at least template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme, and then dispense the first PCR reaction solution into a first 96-well PCR plate. The first 96-well PCR plate is used to contain the first PCR reaction solution, so that N1 PCR cycles are performed in the first PCR reaction solution to obtain the first stage solution; A deep-well plate is used to contain the first-stage solution, and the deep-well plate is also configured with target magnetic beads so that the target magnetic beads bind to the target DNA product to form a carrier conjugate. A 96-well magnetic rack is used to separate the carrier conjugate from the first-stage solution; The deep well plate is also used to add a second PCR reaction solution containing at least primers, dNTPs, DNA polymerase and mismatch recognition enzyme, and to elute the vector conjugate in the second PCR reaction solution to release the target DNA product. The target solid-phase vector after releasing the target DNA product is separated from the second PCR reaction solution to obtain a second-stage solution. The second 96-well PCR plate is used to contain the second stage solution, so that N2 PCR cycles are performed in the second stage solution to obtain an amplification solution containing the target DNA product. Wherein, N1 is a preset first cycle number, and N2 is a preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, and CEL I nuclease.

[0007] A fourth aspect of this invention discloses a method for preparing mRNA, the method comprising: A target DNA template is prepared by any of the DNA amplification methods according to the first aspect of the present invention, wherein the target DNA template includes a target gene coding sequence CDS, and further includes one of a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence, and an SP6 RNA polymerase promoter sequence; The target DNA template was used to perform an in vitro transcription reaction to obtain the target mRNA.

[0008] As an optional implementation, in a fourth aspect of the present invention, the step of using the target DNA template to perform an in vitro transcription reaction to obtain the target mRNA includes: During the in vitro transcription reaction using the target DNA template, a Cap0 or Cap1 structure is introduced at the 5' end of the mRNA using co-transcriptional capping or post-transcriptional capping. A poly(A) tail of 50-250 nucleotides is then added to the 3' end of the mRNA using Poly(A) polymerase to obtain the target mRNA.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The core of this invention lies in a "three-in-one" synergistic strategy: time segmentation: PCR is divided into two stages, with intervention in the middle stage (e.g., cycle 18); spatial separation: impurities and inhibitors are physically removed by selective binding of solid-phase carriers (e.g., magnetic beads); dual-enzyme synergy: DNA polymerase (e.g., 3' mismatch proofreading) + mismatch recognition enzyme (e.g., T7EI for internal mismatch recognition) forms a "double insurance".

[0010] The present invention has found that: Using high-fidelity enzymes alone: ​​the error rate is still 1×10⁻⁶-5 Adding T7EI alone: ​​actually inhibits the accumulation of cleavage products; separating with magnetic beads alone: ​​only improves purity and yield, with limited improvement on error rate; This invention combines the three elements at an appropriate time point (e.g., the 18th cycle, when the product has reached 10% of the initial template). 5 The method involves magnetic bead separation and substrate resetting (which can achieve a comprehensive improvement in low error rate, yield, and purity) to reduce mismatch products (but the products have not yet accumulated exponentially). Specifically, the beneficial effects of the DNA amplification method in this invention include at least the following: (1) High fidelity: error rate less than 5×10 -7 (2) High yield: The yield per reaction reaches 10-15 μg; (3) High purity: Primer residue <1%, protein residue <30 ng / μg; (4) Stable process: Batch-to-batch variation CV <10%; (5) Cost-effective advantages: ① Low unit yield cost: Although the reagent cost per reaction is slightly higher than that of traditional PCR, the overall cost per unit yield (yuan / μg) is reduced by 30-40% due to the yield increase of about 2.5-3 times; ② Reduced repeated experiments: Traditional PCR usually requires 3-4 repetitions to obtain enough DNA template, while the present invention can meet the downstream application needs with a single reaction, saving the time and reagent consumption of repeated experiments; ③ Time cost advantage: Compared with plasmid amplification methods (usually requiring 24-48 hours), the present invention can be completed in 4-5 hours, shortening the time by more than 80% and significantly reducing labor costs. (6) Good automation compatibility: The method of the present invention can be adapted to 96-well / 384-well high-throughput platforms, and the marginal cost is greatly reduced when scaled up. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 This is a schematic flowchart of a DNA amplification method disclosed in an embodiment of the present invention; Figure 2 This is a schematic diagram of the structure of a DNA amplification device disclosed in an embodiment of the present invention; Figure 3 This is a schematic diagram of the structure of a DNA amplification system disclosed in an embodiment of the present invention. Detailed Implementation

[0013] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] The various aspects of the present invention will be described in detail below. Unless otherwise specified, all raw materials of the present invention can be prepared by conventional methods in the art or are commercially available.

[0015] Example 1 Explanation of the invention concept: Given that existing polymerase chain reaction (PCR) technologies are limited by the accumulation of inhibitors and mismatch extension in the reaction system, it is difficult to achieve high-fidelity, high-purity and microgram-milligram level outputs simultaneously within a conventional volume.

[0016] The main inventive concept of this invention lies in: online capture-release (solid-phase cycling selection): at a selected stage of one or more thermal cycles, the reaction mixture is briefly contacted with a solid-phase carrier selective for the target double-stranded DNA and reversibly released to dynamically remove free primers, short mismatches, and other inhibitors during the generation stage, and to enrich / recover the target product for subsequent cycles or collection segments. Dual-enzyme synergistic mismatch inhibition: a first enzyme (with polymerization and proofreading activities) and a second enzyme (with recognition, inhibition, or excision functions for mismatches / non-canonical ends) coexist in the system. Through activity gating and temporal synergy, the accumulation and enrichment of mismatches before they become effective extension templates are inhibited.

[0017] Among them, "online capture-release" refers to the process of contacting the reaction mixture with a solid phase with reversible selectivity and triggering release during the PCR thermal cycling process within a preset time window.

[0018] "Dual-enzyme synergistic mismatch inhibition" refers to the synergistic inhibition of mismatch extension by the first enzyme (with polymerization and proofreading activities) and the second enzyme (with mismatch recognition / inhibition / removal functions) in the same reaction system through the synergistic effects of activity and timing.

[0019] "High purity / high fidelity": refers to the product meeting the preset thresholds for residue, structure, and error rate under specified measurement methods.

[0020] Based on the above inventive concept, Embodiment 1 of the present invention discloses a DNA amplification method, such as... Figure 1 As shown, the method includes: S1. Perform N1 PCR cycles in a first PCR reaction solution containing at least template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme to obtain the first stage solution; S2. Contact the first-stage solution with the target solid-phase support to allow the target solid-phase support to bind with the target DNA product to form a carrier conjugate; separate the carrier conjugate from the first-stage solution; S3. The vector conjugate is eluted in a second PCR reaction solution containing at least primers, dNTPs, DNA polymerase and mismatch recognition enzyme to release the target DNA product. The target solid-phase vector after releasing the target DNA product is separated from the second PCR reaction solution to obtain the second stage solution. S4. Perform N2 PCR cycles in the second-stage solution to obtain an amplification solution containing the target DNA product; In this embodiment of the invention, N1 is a preset first cycle number, and N2 is a preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, CEL I nuclease, EndoMS enzyme, and Surveyor enzyme.

[0021] Optionally, performing N2 PCR cycles in the second-stage solution may include an isothermal amplification step, using an isothermal amplification enzyme.

[0022] In this embodiment of the invention, the solid-phase support can be selected from materials that exhibit selectivity for the length / structure / sequence characteristics of double-stranded DNA, including but not limited to: functionalized inorganic materials, polymer-based microbeads, surface-modified porous substrates, or bioligand-mediated composite materials. The triggered release mechanism can be based on non-destructive triggering methods such as ionic strength, competitive ligands, pH window, or mild temperature changes. Optionally, the support surface chemistry possesses anti-nonspecific adsorption properties to reduce nonspecific binding of enzymes to short oligonucleotides, thereby improving cycling stability and reusability.

[0023] In this embodiment of the invention, the synergistic effect of the two enzymes means: The first enzyme (DNA polymerase) possesses polymerization and proofreading activities, enabling high-fidelity primer extension and correction of partial erroneous incorporation. The second enzyme (mismatch recognition enzyme) has the function of recognizing, inhibiting, or excising mismatched base pairs or non-canonical 3′ ends, raising the threshold for mismatches as extension templates. The second enzyme may possess one or more functional characteristics such as exonucleation, end-modification recognition, or mismatch binding inhibition. The synergistic effect is achieved through timing and activity window: reducing the effective extension probability early in mismatch formation, thereby reducing the final error rate and mismatch enrichment at the system level.

[0024] The core innovation of this invention lies in its "three-in-one" synergistic strategy: Time segmentation: PCR is divided into two stages, with intervention in the middle stage (e.g., cycle 18); Spatial separation: Impurities and inhibitors are physically removed by selective binding of solid-phase carriers (e.g., magnetic beads); Dual enzyme synergy: DNA polymerase (e.g., 3' mismatch proofreading) + mismatch recognition enzyme (e.g., T7EI for internal mismatch recognition) form a "double insurance".

[0025] The embodiments of this invention found that: using high-fidelity enzymes alone still resulted in an error rate of 1×10⁻⁶. -5 Adding T7EI alone: ​​actually inhibits the accumulation of cleavage products; separating with magnetic beads alone: ​​only improves purity and yield, with limited improvement on error rate; This invention combines the three elements at an appropriate time point (e.g., the 18th cycle, when the product has reached 10% of the initial template). 5 Only by performing magnetic bead separation and substrate resetting (which can achieve a comprehensive improvement in low error rate, yield, and purity) can we realize the goal of reducing the error rate, yield, and purity.

[0026] Specifically, the beneficial effects of the DNA amplification method in this invention include at least the following: (1) High fidelity: error rate less than 5×10 -7 (2) High yield: The yield per reaction reaches 10-15 μg; (3) High purity: Primer residue <1%, protein residue <30 ng / μg; (4) Stable process: Batch-to-batch variation CV <10%; (5) Cost-effective advantages: ① Low unit yield cost: Although the reagent cost per reaction is slightly higher than that of traditional PCR, the overall cost per unit yield (yuan / μg) is reduced by 30-40% due to the yield increase of about 2.5-3 times; ② Reduced repeated experiments: Traditional PCR usually requires 3-4 repetitions to obtain enough DNA template, while the present invention can meet the downstream application needs with a single reaction, saving the time and reagent consumption of repeated experiments; ③ Time cost advantage: Compared with plasmid amplification methods (usually requiring 24-48 hours), the present invention can be completed in 4-5 hours, shortening the time by more than 80%, significantly reducing labor costs. (6) Good automation compatibility: The method of the present invention can be adapted to 96-well / 384-well high-throughput platforms, and the marginal cost is greatly reduced when scaled up. In an optional embodiment, the mismatch recognition enzyme is T7 endonuclease I; wherein: the concentration of T7 endonuclease I in the first PCR reaction solution is 0.01-0.1 U / μL; and the concentration of T7 endonuclease I in the second stage solution is 0.005-0.05 U / μL.

[0027] In another optional embodiment, the concentration of T7 endonuclease I in the first PCR reaction solution is 0.025-0.075 U / μL; the concentration of T7 endonuclease I in the second stage solution is 0.0125-0.0375 U / μL.

[0028] In another optional embodiment, the DNA polymerase has 3'→5' exonuclease activity, and the DNA polymerase is at least one of Phusion DNA polymerase, Q5 DNA polymerase, PrimeSTAR DNA polymerase, and Pfu DNA polymerase.

[0029] In yet another alternative embodiment, N1 is set to 10-25 and N2 is set to 10-25.

[0030] In yet another alternative embodiment, N1 is set to 16-19, N2 is set to 13-18, and N1+N2 is set to 30-37.

[0031] In yet another alternative embodiment, N1 is set to 18, N2 is set to 15-17, and N1+N2 is set to 33-35.

[0032] In yet another optional embodiment, the target solid support is hydroxyl-modified magnetic beads, and step S2 specifically includes: The first-stage solution was mixed with the target solid-phase carrier in a PEG-salt solution to bind the target solid-phase carrier with the target DNA product to form a carrier conjugate. The carrier conjugate was obtained by magnetic separation and then washed with ethanol.

[0033] In yet another optional embodiment, the PEG-salt solution consists of 15%-22% w / v polyethylene glycol and 2.0-3.0 M NaCl.

[0034] In yet another optional embodiment, the second PCR reaction solution comprises at least the following components: dNTPs: 150-400 μM dATP, 150-400 μM dGTP, 150-400 μM dTTP, and 150-400 μM dCTP; Primers: 0.1-0.5 μM forward primer and 0.1-0.5 μM reverse primer; DNA polymerase: 0.005-0.02 U / μL; Mismatch recognition enzyme: 0.005-0.05 U / μL; Mg²⁺: 1.0-2.0 mM + .

[0035] In yet another optional embodiment, the second PCR reaction solution comprises at least the following components: dNTPs: 200-300 μM dATP, 200-300 μM dGTP, 200-300 μM dTTP, and 200-300 μM dCTP; Primers: 0.2-0.3 μM forward primer and 0.2-0.3 μM reverse primer; 0.01 U / μL DNA polymerase; 0.005-0.05 U / μL mismatch recognition enzyme; 1.0-2.0 mM Mg²⁺ + .

[0036] Based on the same inventive concept, Embodiment 1 of the present invention also discloses a DNA amplification device, such as... Figure 2 As shown, the device includes: The first reaction chamber 101 contains at least a first PCR reaction solution comprising template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme; the first reaction chamber 101 is used to control the first PCR reaction solution to undergo N1 PCR cycles to obtain the first stage solution. Purification device 102 is connected to first reaction chamber 101. The first stage solution and the target solid-phase carrier are contacted in purification device 102 so that the target solid-phase carrier and the target DNA product are combined to form a carrier conjugate. Purification device 102 is used to separate the carrier conjugate from the first stage solution. The second reaction chamber 103 contains at least a second PCR reaction solution including primers, dNTPs, DNA polymerase, and mismatch recognition enzyme. The second reaction chamber 103 is used to elute the vector conjugate in the second PCR reaction solution to release the target DNA product. The second reaction chamber 103 is also used to separate the target solid-phase vector after the target DNA product has been released from the second PCR reaction solution to obtain the second stage solution. The second-stage solution undergoes N2 PCR cycles in the second reaction chamber 103 to obtain an amplification solution containing the target DNA product. Wherein, N1 is the preset first cycle number, and N2 is the preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, and CEL I nuclease.

[0037] Example 2 Polymerase chain reaction (PCR) is one of the most fundamental and important techniques in molecular biology. However, traditional PCR methods suffer from the following technical bottlenecks: 1. Error rate accumulation problem Standard Taq DNA polymerase lacks 3'→5' exonuclease proofreading activity, with an error rate of approximately 1×10⁻⁶. -5 Up to 1×10 -4 / bp / doubling. Even with the use of high-fidelity DNA polymerases with proofreading activity (such as Phusion, Q5), the error rate is still 1×10⁻⁶. -6 Up to 1×10 -7 Within this range, as the number of PCR cycles increases, errors accumulate exponentially, resulting in a large number of mutants in the final product. This poses a serious problem for the following applications: mRNA drugs and vaccines: Errors in the DNA template can be directly transcribed into mRNA, leading to protein translation errors, increased immunogenicity, or reduced efficacy. COVID-19 mRNA vaccines approved between 2020 and 2023 (such as BNT162b2 and mRNA-1273) have extremely stringent requirements for DNA template quality, with an error rate controlled to within 5 × 10⁻⁶. -7 the following.

[0038] Gene therapy vectors: The construction of vectors such as AAV and lentiviruses requires a mutation-free DNA template. A single nucleotide error may lead to treatment failure or safety issues.

[0039] Synthetic biology: The design of gene circuits and metabolic pathways requires precise DNA sequences.

[0040] 2. Production plateau issue Standard PCR reactions plateau after 25-30 cycles, with yield stagnating. The main reasons include: substrate depletion: 60-80% of dNTPs are consumed in the first 20-25 cycles, with the concentration dropping from the initial 200 μM to below 50 μM, becoming a rate-limiting factor; and inhibitor accumulation: pyrophosphate (PP)... i When the product of DNA polymerase accumulates to >1 mM, it inhibits polymerase; product inhibition: high concentrations of product (>50 nM) preferentially undergo reannealing (product - product Kd ≈ 10). -9 M), rather than binding to the primer (product-primer Kd ≈ 10). -7 M), which causes the amplification efficiency to drop from 85-95% in the early stage to 50-60% in the later stage; enzyme activity decay: DNA polymerase is gradually inactivated during repeated thermal cycling.

[0041] This results in a standard 50-100 μL PCR reaction yielding only 3-6 μg, which is insufficient to meet the needs of downstream applications (such as IVT, NGS library preparation, and cloning). This often requires repeated experiments or larger reaction volumes, increasing costs and time.

[0042] 3. Problem of residual impurities Traditional PCR products contain: primer residues (5-15%), affecting downstream enzyme digestion, sequencing, and IVT; primer dimers (significant at primer concentrations >0.5 μM, consuming dNTPs and generating background); protein residues (DNA polymerase, BSA, etc., at 50-200 ng / μg DNA residues, introducing non-specific products in IVT); and short fragments and mismatch products (5-20% byproducts from non-specific amplification, premature termination, etc.). These impurities remain significantly after conventional column purification or ethanol precipitation, severely impacting DNA quality.

[0043] 4. Limitations of existing technology (1) Limitations of high-fidelity polymerase Existing commercially available high-fidelity DNA polymerases (Phusion, Q5, PrimeSTAR, etc.) reduce the error rate to 1-2 × 10⁻⁶ by enhancing 3'→5' exonuclease activity. -6 However, its proofreading mechanism has inherent limitations: it can only recognize 3' end mismatches (the last 1-2 nucleotides incorporated); it is powerless against mismatches within the DNA strand; and at high dNTP concentrations, the proofreading activity is "submerged" (Km effect).

[0044] (2) Limitations of the additive method CN101545009A discloses the addition of betaine, DMSO, etc. to improve PCR specificity, but the improvement on error rate is limited (reduction <30%), and it may inhibit polymerase activity.

[0045] (3) Limitations of multiple purification methods US8,470,996B2 discloses the insertion of purification steps at different stages of PCR, but column purification has the following problems: low recovery rate (70-85%), resulting in yield loss; inability to remove small molecule inhibitors (such as pyrophosphate); and cumbersome operation, making it difficult to automate.

[0046] (4) Limitations of digital PCR / emulsion PCR Competition is reduced by separating the reaction into microdroplets, but the equipment is expensive and the yield is extremely low (ng-level), making it unsuitable for applications requiring μg-level products.

[0047] 5. Specific requirements of DNA template for in vitro mRNA transcription The rapid development of mRNA drugs and vaccines (the global market size exceeded US$50 billion in 2021 and is expected to reach US$200 billion by 2030) has placed extremely stringent requirements on DNA templates: Ultra-high purity: protein residue <10 ng / μg, primer residue <0.5%, otherwise a large amount of dsRNA byproducts will be generated in IVT (triggering innate immunity and reducing translation efficiency); Ultra-low error rate: DNA template errors will result in 100% transcription to mRNA, which will then be translated into the incorrect protein. For therapeutic mRNAs of 500-2000 nt, a DNA template error rate of <5×10⁻⁶ is required. -7 ; High yield: Clinical-grade mRNA production requires mg-g levels, corresponding to hundreds of μg to tens of mg of DNA template; High integrity: Truncation of DNA template produces truncated mRNA, which reduces efficacy and may trigger an immune response; Batch stability: GMP production requires batch-to-batch variation CV < 10%.

[0048] Currently, the industrial production of IVT DNA templates mainly relies on plasmid amplification or gene synthesis, but this method suffers from problems such as long cycle time (48-72 hours), high cost, and limited modification options. PCR template preparation has advantages such as speed (<5 hours), flexibility (easy to introduce modifications), and low cost, but it has not yet become mainstream due to the aforementioned quality issues.

[0049] 6. The Potential and Challenges of Mismatch Detection Technology T7 endonuclease I (T7EI) is a mismatch recognition enzyme that specifically recognizes and cleaves mismatches, insertions / deletions, heteroduplexes, and cross-shaped structures in DNA double strands. Its mechanism of action is as follows: Scan the DNA double strands to identify non-standard Watson-Crick base pairs; cleave both strands at mismatch sites ±1-3 bp; release short fragments and 3'-OH ends.

[0050] In theory, T7EI can eliminate mismatch products in PCR, but direct addition presents problems: high concentrations of T7EI (>0.2 U / μL) cleave normal DNA products (Mg²⁺). + The activity of nonspecific nucleases depends on the concentration; low concentrations (<0.01 U / μL) are not efficient at clearing mismatches; the accumulation of cleavage products actually acts as an inhibitor.

[0051] Therefore, the key to improving PCR fidelity is to make reasonable use of T7EI or similar mismatch recognition enzymes to remove mismatch products without affecting normal amplification.

[0052] In summary, existing PCR technologies struggle to simultaneously meet the requirements of high fidelity, high yield, and high purity, severely limiting their application in high-end applications such as in vitro mRNA transcription. There is an urgent need to develop new PCR strategies to overcome these bottlenecks.

[0053] The purpose of this invention is to address the shortcomings of existing PCR technologies in terms of fidelity, yield, and purity by providing a high-fidelity, high-yield DNA amplification method and its application in in vitro mRNA transcription, thereby solving at least one of the aforementioned problems in the prior art.

[0054] To achieve the above objectives, the present invention discloses the following technical solution, the overall process of which is as follows: Template DNA: Phase 1: Early amplification (cycles 1-18) + high-fidelity enzyme + T7EI; Phase 2: Separate magnetic beads, remove inhibitors, and replace with fresh reagents; Phase 3: Late-stage amplification (cycles 19-35), dNTP reset; ->High-fidelity, high-yield DNA->IVT template / direct application Specifically, a high-fidelity, high-yield DNA amplification method includes the following steps: (1) First-stage amplification (early amplification): Amplification was performed for 15-20 cycles in a PCR reaction system containing template DNA, primers, high-fidelity DNA polymerase, dNTPs, and mismatch recognition enzyme.

[0055] Template DNA: This can be genomic DNA, plasmid DNA, cDNA, or synthetic DNA, with a copy number of 10² to 10⁻⁶. 8 10 preferred 4 Up to 10 6 copy; High-fidelity DNA polymerase: with 3'→5' exonuclease activity, such as Phusion, Q5, PrimeSTAR or Pfu DNA polymerase, at a concentration of 0.01-0.03 U / μL; dNTPs: The concentration of each dNTP (dATP, dCTP, dGTP, dTTP) is 150-400 μM, preferably 200-300 μM; Primers: 0.2-0.8 μM each for forward and reverse primers, preferably 0.5 μM each; Mismatch recognition enzymes: T7 endonuclease I (concentration 0.01-0.1 U / μL, preferably 0.05 U / μL), MutS protein (0.1-1 μM) or CEL I nuclease (0.01-0.1 U / μL); Buffer solution: containing Mg²⁺ + PCR buffer, Mg² + The concentration is adjusted according to the total dNTP concentration to ensure free Mg²⁺. +Maintain at 1.5-2.5 mM; reaction volume: 50-200 μL, preferably 100 μL; cycling parameters: initial denaturation: 95-98°C, 20-60 seconds; cycles (15-20): denaturation: 95-98°C, 5-15 seconds; annealing: set according to primer Tm, usually 55-65°C, 15-30 seconds; extension: 68-72°C, 15-60 seconds / kb; after completion, maintain at 4°C for further processing.

[0056] Key point 1: Synergistic effect of low concentration of T7EI Adding a low concentration of T7EI (0.01-0.1 U / μL) to the PCR system utilizes its mismatch recognition capability to identify newly generated mismatches during the annealing-extension phase of each cycle. T7EI cleaves DNA at the mismatch site, producing short fragments with 3'-OH. These fragments: cannot serve as efficient amplification templates (<50 bp); are diluted in subsequent cycles; and prevent exponential amplification of mismatch products.

[0057] Meanwhile, low concentrations of T7EI exhibit extremely low (<1%) cleavage activity against correctly paired DNA double strands, without affecting the amplification of normal products. The 3'→5' exonuclease activity of the high-fidelity polymerase and the mismatch recognition ability of T7EI form a "double insurance," reducing the error rate by 22 times.

[0058] (2) Intermediate purification (magnetic bead separation and washing): After the first stage of amplification is completed, the following purification steps are performed: a. DNA binding to magnetic beads: Transfer the first-stage amplification product to a centrifuge tube; add 1.2-2.0 times the volume (relative to the reaction volume) of PEG-salt solution, wherein the PEG-salt solution consists of 15-22% w / v polyethylene glycol (PEG, molecular weight 6000-10000 Da, preferably PEG8000) and 2.0-3.0 M NaCl; after mixing, add carboxyl-modified magnetic beads (concentration 8-15 mg / mL, amount 1.0-1.5 times the reaction volume, preferably 1.2 times); incubate at room temperature for 3-8 minutes to allow DNA to bind to the surface of the magnetic beads through ionic bonds; b. Magnetic separation: Place the centrifuge tube on a magnetic rack and let it stand for 1-3 minutes until the magnetic beads aggregate and the supernatant becomes clear; carefully aspirate the supernatant (containing primers, primer dimers, dNTPs residues, pyrophosphate, mismatch products, proteins, etc.). c. Ethanol washing: Place the tube on the magnetic rack, add 70-85% v / v ethanol (1.5-2.5 times the volume of the magnetic beads), and let stand for 30-60 seconds; remove the ethanol; repeat the washing 1-2 times (2-3 washes in total); after the last wash, carefully remove any remaining ethanol. d. Dry air: Open the tube cap and allow it to dry at room temperature for 3-6 minutes (preferably 3-5 minutes) until the surface of the magnetic beads is dull but not cracked. Key Point 2: Advantages of Magnetic Bead Separation Compared to traditional column purification, magnetic bead separation has the following advantages: High recovery rate: >90% (column purification only 70-85%); High selectivity: By adjusting the PEG concentration, it selectively binds to DNA fragments of the target size (>200 bp), removing primers (20-30 bp) and short fragments (<100 bp); Removal of small molecule inhibitors: Ethanol washing effectively removes pyrophosphate, excess dNTPs, salts, etc.; Protein removal: Proteins such as DNA polymerase and BSA do not bind to magnetic beads and are removed in the supernatant and washing solution; Easy automation: Magnetic separation can be automated with high throughput in 96-well or 384-well systems using a liquid handling workstation 201.

[0059] (3) Change the reaction system (elution and substrate reset): The DNA product bound to the magnetic beads was eluted in a reaction mixture containing fresh dNTPs, primers, high-fidelity DNA polymerase, mismatch recognition enzyme, and buffer. Specific steps: a. Prepare the second PCR mixture: The composition is similar to the first stage, but the concentration of each component can be adjusted: dNTPs: 150-400 μM each (preferably 200-300 μM), restored to the initial level; primers: 0.1-0.5 μM each (can be halved to 0.2-0.3 μM, as the primers from the first stage were not completely consumed); high-fidelity DNA polymerase: 0.005-0.02 U / μL (can be halved, as the enzyme still has residual activity from the first stage); T7EI: 0.005-0.05 U / μL (can also be halved); buffer and Mg²⁺ + Adjust according to the new dNTP concentration; do not contain template DNA (the DNA eluted from the magnetic beads is the template); volume: 0.5-1.5 times the volume of the first stage reaction, preferably equal (50-200 μL); b. DNA elution: Add the above second PCR mixture to the dried magnetic beads; vortex or invert to mix thoroughly, so that the magnetic beads are completely resuspended; place the centrifuge tube in a 65-75°C (preferably 70°C) heat block or PCR instrument and incubate for 2-5 minutes (preferably 3 minutes) to break the ionic bonds between DNA and magnetic beads; briefly vortex and then centrifuge. c. Separate the magnetic beads and recover the eluent: Place the tube on a magnetic rack and separate for 1-3 minutes; carefully aspirate the supernatant (containing the eluted DNA and fresh reaction mixture); transfer to a new PCR tube and add nuclease-free water to the predetermined volume (e.g., 100 μL). Key Point 3: The Significance of Substrate Resetting By replacing the reaction mixture with a completely new one, the following can be achieved: dNTP concentration recovery: from 50-80 μM at the end of the first stage to 200-300 μM, eliminating substrate limitation; removal of inhibitors: pyrophosphate concentration reduced from 1-2 mM to <0.01 mM, restoring polymerase activity; removal of mismatch products: mismatch products generated in the first stage (which have been cleaved into short fragments by T7EI) are removed during magnetic bead separation and will not continue to amplify in the second stage; primer concentration adjustment: primer concentration is appropriately reduced (because there are already products as templates), reducing primer dimer formation. This is equivalent to "restarting" the PCR reaction, extending the efficient amplification period, and breaking through the plateau limitation.

[0060] (4) Second-stage amplification (later amplification): The eluted DNA product is amplified for 12-20 cycles (preferably 15-17 cycles) to obtain high-fidelity, high-yield DNA product. Cycling parameters: same as the first stage (denaturation, annealing, extension temperature and time); final extension: 72°C, 3-10 minutes (preferably 5 minutes) to ensure complete extension of all DNA strands; storage: short-term storage at 4°C, or long-term storage at -20°C / -80°C; Key point 4: Optimization of total number of loops The first stage consists of 18 cycles, and the second stage consists of 17 cycles, totaling 35 cycles. Compared to the 30 cycles of traditional PCR, this is only an increase of 5 cycles, but because: each stage maintains high efficiency (85-95%); mismatch products and inhibitors are removed in the middle; there is sufficient substrate and no plateau phase limitation; the yield is increased by 2.5-3 times, while the error rate is reduced (by 22 times).

[0061] Technical effects: Through the above technical solution, the DNA product obtained by this invention has the following excellent properties: 1. Production increased significantly. For a 1 kb fragment, a 100 μL reaction system can yield 10-15 μg of DNA (compared to 3-5 μg for conventional PCR). For fragments ranging from 500 bp to 2 kb, the yield is increased by 2.5 to 3 times; For 3-5 kb long fragments, the yield is increased by 2-2.5 times; 2. Error rate significantly reduced Validated by high-throughput sequencing, the error rate is <5×10⁻⁶. -7 (Traditional high-fidelity PCR is 1-1.5×10⁻⁶)-5 Compared to using high-fidelity polymerase alone, the error rate is reduced by 22 times; compared to adding T7EI (non-magnetic bead separation) alone, the error rate is reduced by 8 times. 3. Extremely high purity Primer residue <1% (5-15% in traditional PCR); protein residue <30 ng / μg DNA (50-200 ng / μg in traditional PCR); short fragments and primer dimers <2% (5-20% in traditional PCR); A 260 / A 280 = 1.80-1.90, A 260 / A 230 = 2.0-2.3; 4. Excellent integrity Agarose gel electrophoresis showed a single bright band without tailing; Bioanalyzer analysis showed that the target fragment accounted for >95%; suitable for direct use in downstream applications without additional purification. 5. Good batch stability The coefficient of variation (CV) of yield in 10 independent trials was <12%; the error rate (CV) was <18%. Suitable for industrial production; 6. Excellent performance in in vitro mRNA transcription As an IVT template, it increased mRNA yield by 59% compared to templates prepared by conventional PCR; mRNA integrity (RIN value) increased from 8.2±0.5 to 9.5±0.2; dsRNA contamination decreased from 2-5% to <0.1%; and translation efficiency increased by 45-60%. 7. Easy to operate and scale up Total time is 4.5 hours (traditional PCR takes 3 hours, but requires multiple repetitions and additional purification); the magnetic bead separation step can be completed within 30 minutes; it is easy to automate and can be scaled up to 96-well or 384-well high-throughput. Summary of the core innovations of the invention The core innovation of this invention lies in the "three-in-one" synergistic strategy: Time segmentation: PCR is divided into two stages, with intervention in the middle stage (cycle 18); Spatial separation: Impurities and inhibitors are physically removed through selective binding of magnetic beads; Dual enzyme synergy: High-fidelity polymerase (corrects 3' mismatches) + T7EI (identifies internal mismatches) form a "double insurance". All three are indispensable: Using high-fidelity enzymes alone: ​​the error rate is still 1×10⁻⁶ -5 Adding T7EI alone: ​​actually inhibits the accumulation of cleavage products; separating with magnetic beads alone: ​​only improves purity and yield, with limited improvement on error rate; Only by combining these three factors at the appropriate time (cycle 18, when the product has reached 10% of the initial template) can we achieve this. 5 Only by performing magnetic bead separation and substrate resetting (which can be done to achieve a comprehensive improvement in error rate, yield, and purity) can the mismatch products be increased (but have not yet accumulated exponentially).

[0062] Example 3 Based on the same concept, Embodiment 3 of the present invention discloses a DNA amplification method, which is described in detail below: Implementation Method 1: Preparation of a 1 kb high-fidelity DNA fragment

Experimental Materials

[0063]

Experimental Procedure

[0064] Mix well, briefly centrifuge, transfer to a PCR instrument, and run the following program:

[0065] Quality control point 1: Take 2 μL of reaction solution, dilute with 10 μL of water, and take 5 μL for 1% agarose gel electrophoresis. A bright 1 kb band should be observed. Qubit quantification: concentration approximately 50-80 ng / μL (total 5-8 μg).

[0066] (2) Magnetic bead separation and washing a. After PCR is complete, remove the PCR tube, centrifuge briefly for 5 seconds, and transfer all 100 μL of reaction solution to a 1.5 mL low-adsorption centrifuge tube.

[0067] b. Add 180 μL Binding Buffer (1.8 × volume), invert and mix 10 times, and let stand at room temperature for 5 minutes.

[0068] c. Add 120 μL of magnetic bead suspension (vortex for 30 seconds before use), invert and mix 20 times, and let stand at room temperature for 5 minutes (the solution will turn a uniform brown color).

[0069] d. Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely gathered on the magnet side and the supernatant is clear.

[0070] e. Carefully aspirate the supernatant (approximately 400 μL) and discard it into the waste tube.

[0071] f. With the tube on the magnetic rack, slowly add 200 μL of freshly prepared 80% ethanol along the tube wall, let stand for 30 seconds, and then discard the ethanol.

[0072] g. Repeat step f once (second ethanol wash).

[0073] h. Carefully aspirate the remaining ethanol with a 10 μL pipette tip, open the cap, and dry at room temperature for 4 minutes (until the surface of the magnetic bead becomes matte and there are no visible droplets).

[0074] Quality control point 2: Take 10 μL of the retained supernatant for gel electrophoresis. There should be no obvious 1 kb band (confirming that the DNA has bound to the magnetic beads).

[0075] (3) Prepare the second PCR mixture and elute the DNA. Prepare 100 μL of the second PCR mixture on ice:

[0076] Add the above mixture to the dried magnetic beads, tighten the lid, vortex for 10 seconds to completely resuspend the magnetic beads, and then centrifuge briefly.

[0077] Place the centrifuge tube in the PCR instrument's heating block or metal bath and incubate at 70°C for 3 minutes. Remove, vortex for 5 seconds, briefly centrifuge, and separate on a magnetic rack for 2 minutes. Carefully aspirate all supernatant (approximately 95-98 μL) and transfer to a new 0.2 mL PCR tube, then add nuclease-free water to a final volume of 100 μL. Quality control point 3: Qubit quantification: The concentration should be 50-100 ng / μL to confirm successful DNA elution. Recovery rate = (eluted DNA amount) / (first-stage DNA amount) = 90-95%.

[0078] (4) Second-stage PCR (cycles 19-35) Place the PCR tube containing the eluted DNA into a PCR instrument and run the following program:

[0079] (5) Product Analysis Take 5 μL of the final product and perform the following tests: a. Agarose gel electrophoresis (1% gel, 120V, 30 minutes): Results: A single bright band at the 1 kb position, no primer dimers (<100 bp), no tailing, and no nonspecific bands.

[0080] b. Qubit dsDNA HS Assay quantification: Concentration: 125±8 ng / μL; Total yield: 12.5±0.8 μg (95μL final volume); c. NanoDrop purity analysis: A 260 / A 280 = 1.86±0.02; A 260 / A 230 = 2.15±0.08; d. Agilent Bioanalyzer analysis: The main peak was located at 1035 bp, accounting for 97.2% of the total area; RIN (RNA Integrity Number) = 9.8; no obvious degradation or short fragments were observed. e. High-throughput sequencing error rate analysis (Illumina NovaSeq): Sequencing depth: 10,000×; Error rate: 4.2×10⁻⁶ -7 (Confidence interval: 3.8-4.6×10) -7 Error type distribution: Replacement errors: 78% (A) G, T C-transformations accounted for 60%; missing: 15%; insertion: 7%; f. Residue analysis: Primer residue (Bioanalyzer peak area): 0.8%; Protein residue (Bradford method): 22 ng / μg DNA; Primer dimer: <0.5%; Implementation Method 2: Control Experiment (Traditional High-Fidelity PCR) Perform standard 30-cycle PCR using the same template, primers, and Phusion polymerase, but without adding T7EI and without mid-cycle magnetic bead separation.

[0081] [Reaction system] (100 μL)

[0082] PCR Procedure

[0083]

Results Analysis

[0084] Implementation Method 3: Control Experiment (T7EI only added, no magnetic beads for separation) Same as in Implementation Method 2, but with the addition of 5 μL T7EI (final concentration 0.05 U / μL) to the reaction system, no intermediate magnetic bead separation, and 30 cycles of continuous PCR.

[0085]

Results Analysis

[0086] Conclusion: Adding T7EI alone can reduce the error rate by approximately 2.7 times, but the accumulation of cut products leads to a decrease in yield, and it cannot remove impurities. T7EI must be combined with magnetic bead separation to fully realize its advantages.

[0087] Implementation Method 4: Control Experiment (Magnetic Bead Separation Only, No T7EI Added) The two-stage PCR and magnetic bead separation process is the same as in Implementation Method 1, but T7EI is not added to the reaction system.

[0088]

Results Analysis

[0089] Conclusion: Magnetic bead separation primarily improves yield and purity (through substrate reset and impurity removal), with limited improvement in error rate (approximately 3-fold). The addition of T7EI further reduces the error rate by 7-8 times (from 3.2 × 10⁻⁶). -6 Reduced to 4.2×10 -7 ), achieving a total reduction of 22 times.

[0090] Implementation Method 5: Verification of Different Segment Lengths The method of this invention was used to amplify DNA fragments of different lengths and to evaluate its versatility.

[0091] [Segment Design]

[0092] Other parameters are the same as in Implementation Method 1, except for the extension time (calculated at 30 seconds / kb).

[0093]

result

[0094] Conclusion: The method of this invention is effective for fragments ranging from 200 bp to 5 kb, increasing yield by 2-3.2 times, with an error rate of <7×10⁻⁶. -7 The improvement factor for long fragments (>3 kb) is slightly lower, but still significantly better than traditional methods.

[0095] Implementation Method 6: Experiment on Optimizing the Number of Cycles The first stage is fixed at 18 cycles, and the number of cycles in the second stage is varied (12-22 times), and the impact on output and error rate is evaluated.

[0096]

result

[0097] Conclusion: The optimal choice is 15-17 cycles in the second stage (33-35 cycles in total), achieving a yield of 12-13 μg and maintaining an error rate of 4-5 × 10⁻⁶. -7 After more than 38 total iterations, the error rate begins to rise significantly.

[0098] Implementation Method 7: Optimization of Magnetic Bead Separation Timing With a fixed total number of cycles of 35, the time points for magnetic bead separation were varied (after cycles 12, 15, 18, 21, and 24) to assess the impact.

[0099]

result

[0100] Conclusion: The 18th cycle is the optimal separation point. At this time: The product has reached 2¹ of the initial template 8 ≈ 2.6 × 10 5 The DNA amount is sufficient (5-8 μg); mismatch products have not yet accumulated exponentially (accounting for <1%); inhibitors (such as pyrophosphate) have begun to accumulate significantly; the dNTP concentration has dropped to 80-100 μM and is about to become a rate-limiting factor.

[0101] Implementation Method 8: T7EI Concentration Optimization With other conditions fixed, the concentration of T7EI in the first stage was varied (0, 0.01, 0.05, 0.1, 0.2 U / μL), and the effects were evaluated.

[0102]

result

[0103] Conclusion: A T7EI concentration of 0.05 U / μL is optimal. At this concentration, T7EI can effectively identify and cleave mismatched products, reducing the error rate from 3.2 × 10⁻⁵ U / μL. -6 Reduced to 4.2×10 -7(Reduced by 7.6 times), while the non-specific cleavage of normal products is negligible. Excessively high concentrations (>0.1 U / μL) will cause cleavage of normal products, resulting in a decrease in yield.

[0104] Implementation Method 9: Applicability of Different High-Fidelity Polymerases The method of this invention was used to test the effectiveness of different commercial high-fidelity DNA polymerases.

[0105] Polymerase type

[0106] The other conditions are the same as in Implementation Method 1, except that the polymerase is replaced.

[0107]

result

[0108] Conclusion: The method of this invention is applicable to various high-fidelity DNA polymerases, and can reduce the error rate by 19-23 times. Phusion polymerase performs best (highest yield, lowest error rate) and is recommended as the first choice.

[0109] Example 4 Embodiment 4 of this invention discloses a method for preparing mRNA, the method comprising: According to any one of the DNA amplification methods of Embodiments 1 to 3 of the present invention, a target DNA template is prepared, wherein the target DNA template includes a target gene coding sequence CDS, and also includes one of the T7 RNA polymerase promoter sequence, the T3 RNA polymerase promoter sequence, and the SP6 RNA polymerase promoter sequence; The target DNA template is used to perform an in vitro transcription reaction to obtain the target mRNA.

[0110] In an optional embodiment, an in vitro transcription reaction is performed using a target DNA template to obtain target mRNA, including: In the in vitro transcription reaction using the target DNA template, a Cap0 or Cap1 structure is introduced at the 5' end of the mRNA by co-transcriptional capping or post-transcriptional capping. Poly(A) polymerase is then used to enzymatically add a polyA tail of 50-250 nucleotides to the 3' end of the mRNA to obtain the target mRNA.

[0111] A comparative experiment on in vitro transcription of mRNA was conducted using high-fidelity DNA fragments prepared in any of Examples 1 to 3 and DNA fragments prepared by conventional PCR as templates.

[0112] DNA template design Construct a DNA template containing the following elements: T7 promoter: 5'-TAATACGACTCACTATAG-3'; 5'UTR: KOZAK sequence + 50 nt optimized UTR; CDS: Firefly Luciferase gene (1650 bp); 3'UTR: 120 nt stabilization sequence; PolyA signal: DNA sequence encoding 120 A's; Total length: approximately 1970 bp; Template preparation

[0113] [In vitro transcription reaction] IVT using the MEGAscript™ T7 Transcription Kit (Invitrogen): Reaction system (20 μL): DNA template: 500 ng (normalized); ATP, CTP, GTP, UTP: 7.5 mM each; 10×Reaction Buffer: 2 μL; T7 RNA polymerase mixture: 2 μL; incubate at 37°C for 4 hours; after the reaction, add 2 μL LDNase I (2 U / μL) and digest at 37°C for 30 minutes.

[0114] Purify mRNA using the MEGAclear™ Transcription Clean-Up Kit.

[0115] [mRNA production comparison]

[0116] Key findings: The DNA template prepared by the method of this invention has a 59% higher mRNA yield than that of conventional PCR (from 30.5 to 48.5 μg). The yield was close to that of the plasmid template (52.2 μg of plasmid, only 7.6% higher, with no statistically significant difference). Possible reason: The template of this invention has high purity and few protein and primer residues, which reduces the inhibition of T7 polymerase.

[0117] [mRNA quality analysis] Agarose gel electrophoresis (1% TBE-formaldehyde denaturing gel):

[0118] Agilent Bioanalyzer (RNA 6000 Nano Kit):

[0119] Key findings: The mRNA transcribed from the template of this invention had a RIN value of 9.52, significantly higher than that of the traditional PCR group (8.21, P<0.001), and close to that of the plasmid group (9.68), with no statistically significant difference; dsRNA contamination was extremely low (<0.1%), far lower than that of the traditional PCR group (2.3%). Analysis of the dsRNA generation mechanism: DNA mismatch in the traditional PCR template (9.5×10⁻⁶) -6 During IVT, some mismatch sites cause T7 polymerase to pause or dissociate; the dissociated RNA strand can serve as a template for reverse transcription (due to the inefficient reverse activity of T7 polymerase), forming dsRNA; the template error rate of this invention is extremely low (4.2 × 10⁻⁶). -7 This reduces such abnormal transcriptional events.

[0120] (3) Sequence fidelity analysis (RNA-seq): The transcribed mRNA was reverse transcribed and sequenced using high-throughput sequencing (10,000× depth), and the differences from the expected sequence were statistically analyzed.

[0121]

[0122] Discovery: Errors in the DNA template are almost 100% transcribed into mRNA. The template of this invention has a low error rate (4.2 × 10⁻⁶). -7 It can be directly converted into high-quality mRNA.

[0123] (4) Capping efficiency analysis: The proportion of Cap0 structures was determined using the Cap-Clip™ Acid Pyrophosphatase method.

[0124]

[0125] Findings: The capping efficiency was slightly higher than that of the traditional PCR group, possibly because the high purity of the template reduced the inhibition of the capping enzyme.

[0126] [Functional Validation: In Vitro Translation Assay] In vitro translation was performed using the Rabbit Reticulocyte Lysate System (Promega), and luciferase activity was detected.

[0127] Reaction system (25 μL): Rabbit Reticulocyte Lysate: 17.5 μL; Amino Acid Mixture (1 mM): 0.5 μL; mRNA template: 500 ng; RNasin® Ribonuclease Inhibitor: 0.5 μL Incubate at 30°C for 90 minutes; after the reaction, take 10 μL, add Luciferase Assay Reagent, and measure the luminescence intensity (RLU).

[0128]

result

[0129] Key findings: The mRNA transcribed from the template of this invention has a translation efficiency that is 60% higher than that of the traditional PCR group; it is close to that of the plasmid group (only 12% lower, with no statistically significant difference); possible mechanisms: low error rate → correct protein sequence → correct folding → high activity; less dsRNA contamination → no activation of PKR kinase → no inhibition of translation initiation; high integrity → no premature ribosome shedding → high proportion of full-length protein.

[0130] [Functional Validation: Cell Transfection Experiment] The intracellular translation efficiency and stability of mRNA were verified using HEK293 cells.

[0131] Experimental design: Cells: HEK293 (ATCC CRL-1573); Transfection reagent: Lipofectamine Messenger MAX (Invitrogen); mRNA dosage: 500 ng / well (24-well plate); Detection time points: 4, 8, 12, 24, and 48 hours; Luciferase activity was measured after cell lysis at each time point.

[0132]

result

[0133] Key findings: At all time points, the translational activity of mRNA in the experimental group was significantly higher than that in the control group 1 (P<0.01). At 48 hours, the mRNA in the experimental group still maintained high activity, suggesting that it had better intracellular stability. Possible mechanism: Less dsRNA contamination does not activate the innate immune system (RIG-I, MDA5 pathway), thus reducing mRNA degradation.

[0134] [Immunogenicity Assessment] The immunogenicity of mRNA was assessed using human peripheral blood mononuclear cells (PBMCs).

[0135] Experimental design: Cells: healthy donor PBMCs (n=3); Transfection: Lipofectamine MessengerMAX; mRNA dosage: 1 μg / mL; incubation time: 24 hours; detection: ELISA to measure cytokines in culture supernatant.

result

[0136] Key findings: The level of type I interferon (IFN-α / β) induced by mRNA in the experimental group was significantly lower than that in the control group 1 (P<0.001). The results showed that the mRNA transcribed from the DNA template prepared by the method of this invention had extremely low immunogenicity and was suitable for therapeutic applications.

[0137] [Summary of Implementation Method 10] The high-fidelity DNA template prepared by the method of this invention is used for in vitro mRNA transcription and has comprehensive advantages over traditional PCR methods:

[0138] These advantages make the method of the present invention particularly suitable for the production of GMP-grade mRNA drugs and vaccines.

[0139] Example 5 Embodiment 5 of the present invention discloses a DNA amplification system, such as Figure 3 As shown, the system may include: Liquid processing workstation 201 is used to prepare a first PCR reaction solution containing at least template DNA, primers, dNTPs, DNA polymerase and mismatch recognition enzyme, and then dispense the first PCR reaction solution into a first 96-well PCR plate 202. The first 96-well PCR plate 202 is used to contain the first PCR reaction solution, so that N1 PCR cycles are performed in the first PCR reaction solution to obtain the first stage solution; Deep well plate 203 is used to contain the first-stage solution. Target magnetic beads are also configured in deep well plate 203 so that the target magnetic beads can bind to the target DNA product to form a carrier conjugate. A 96-well magnetic rack 204 is used to separate the carrier conjugate from the first-stage solution; The deep well plate 203 is also used to add a second PCR reaction solution containing at least primers, dNTPs, DNA polymerase and mismatch recognition enzyme, and to elute the vector conjugate in the second PCR reaction solution to release the target DNA product. The target solid-phase vector after releasing the target DNA product is separated from the second PCR reaction solution to obtain the second stage solution. The second 96-well PCR plate 205 is used to contain the second-stage solution, so that N2 PCR cycles can be performed in the second-stage solution to obtain an amplification solution containing the target DNA product. Wherein, N1 is the preset first cycle number, and N2 is the preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, and CEL I nuclease.

[0140] Implementation Method 11: Large-Scale Validation (96-well High-Throughput) To verify the industrial feasibility of the method of the present invention, a large-scale experiment was conducted using a 96-well plate and an automated liquid handling workstation 201.

[0141] Equipment and Materials 96-well PCR plate (0.2 mL / well); 96-well deep-well plate 203 (2 mL / well, for magnetic bead separation); 96-well magnetic rack 204 (Alpaqua); Liquid handling workstation 201 (Hamilton STAR); 96-well PCR instrument (Bio-Rad CFX96). Experimental Design Simultaneously process 96 samples (using the same template to evaluate intra-batch reproducibility); reaction volume per well: 100 μL Other parameters are the same as in Implementation Method 1; the magnetic bead separation step is automatically completed by the liquid handling workstation 201. [Automated Processes] The liquid processing workstation 201 prepares the first PCR mixture and dispenses it into 96-well PCR plates; the PCR instrument performs the first stage of amplification (18 cycles); the mixture is automatically transferred to deep well plate 203; binding buffer and magnetic beads are automatically added; the 96-well magnetic rack 204 performs magnetic separation; the supernatant is automatically aspirated and discarded; the mixture is automatically washed with ethanol ×2; the second PCR mixture is automatically added and eluted at 70°C; the mixture is then transferred to a new 96-well PCR plate. Second-stage PCR amplification (17 cycles); automatic sampling to the detection plate, and automatic quantification using Qubit Flex.

[0142]

result

[0143] in conclusion: The method of this invention is perfectly suited for high-throughput automation; Excellent intra-batch repeatability (CV<10%), meeting industrial production standards; after automation, the total time for 96 samples is only 5.5 hours, and the manual time is less than 1 hour.

[0144] Implementation Method 12: Stability Verification The stability of the DNA products prepared by the method of the present invention under different storage conditions was evaluated.

[0145] Experimental Design The DNA product prepared using Embodiment 1 was divided into several equal portions and stored at: 4°C (refrigerator); -20°C (normal refrigerator); -80°C (ultra-low temperature refrigerator); room temperature (25°C, as extreme conditions); and samples were taken for testing on days 0, 7, 14, 30, 60, and 90.

[0146] [Detection Indicators] Concentration (Qubit); integrity (agarose gel electrophoresis); sequence fidelity (Sanger sequencing, randomly selecting a 200bp region); IVT activity (transcribed mRNA and quantified);

result

[0147] Integrity (agarose gel electrophoresis, percentage of main band)

[0148] IVT activity (relative to mRNA yield, %)

[0149] Recommended storage conditions: Optimal: -80°C, stable for >1 year; Standard: -20°C, suitable for routine laboratories, 6-12 months; Short-term: 4°C, suitable for frequently used samples, 1-2 months; Prohibited: Room temperature storage (significant degradation even in short periods). Additional recommendations: Avoid repeated freeze-thaw cycles (each freeze-thaw cycle results in a 5-10% loss of activity); aliquot storage is recommended (10-20 μL / tube); adding EDTA (final concentration 1 mM) can inhibit nucleases; store protected from light (UV light can cause pyrimidine dimerization).

[0150] Implementation Method 13: Comparison of Different Mismatch Recognition Enzymes In addition to T7EI, the effects of other mismatch recognition enzymes in the method of this invention were tested.

[0151] [Enzyme Type]

[0152] Experimental Design Using the conditions of Implementation Method 1, only the mismatch recognition enzymes were changed: T7EI: 0.05 U / μL; MutS: 0.5 μM (no cleavage, only labeling mismatches to prevent their amplification); CEL I: 0.05 U / μL; Surveyor: 0.05 U / μL; Control: no enzyme added;

result

[0153] in conclusion: T7EI performs best overall: it has the lowest error rate and no non-specific cleavage; MutS only recognizes but does not cleave, so its effect is limited; CEL I and Surveyor are close to T7EI in performance, but they have slight non-specific cleavage and are expensive; T7EI is recommended as the first choice for mismatch recognition enzyme.

[0154] Implementation Method 15: GMP-level Production Validation In accordance with GMP (Good Manufacturing Practice) requirements, verify the controllability and batch consistency of the method of this invention.

[0155] GMP Requirements The batch-to-batch variability of the active pharmaceutical ingredient (DNA template) must be less than 15%; the critical quality attribute (CQA) must have a clear acceptance standard; process validation is required, with at least three consecutive batches passing the test.

[0156]

Critical Control Points (CPP) of Process Parameters

[0157] Key Quality Attributes (CQA) and Acceptance Criteria

[0158] [Process Validation: 3 batches produced] Each batch produces 96 samples (96-well plates), for a total of 3 batches (batch A, B, and C).

[0159] Batch consistency results:

[0160] Intra-batch consistency (96 samples per batch):

[0161] Pass rate statistics:

[0162] in conclusion: All three batches of production validation passed; The batch-to-batch variation CV is <13%, which meets GMP requirements (<15%); the batch-to-batch variation CV is <11%, indicating a highly controllable process; the pass rate is >96%, which meets industrial production standards; the method of this invention is suitable for GMP-level large-scale production.

[0163] In summary, the embodiments of the present invention have the following significant advantages over the prior art: 1. Error rate significantly reduced (22 times) Error rate of the method of this invention: 4.2 × 10⁻⁶ -7 The error rate of traditional high-fidelity PCR is 9.5 × 10⁻⁶. -6 ; Reduced by 22.6 times, achieving plasmid-level accuracy; Technological breakthrough: Through a "three-in-one" strategy of high-fidelity polymerase (correcting 3' mismatches) + T7EI (identifying internal mismatches) + mid-stage magnetic bead separation (removing mismatch products), it is the first time in the PCR field that <5×10 -7 Extremely low error rate.

[0164] Application value: It meets the stringent requirements of mRNA drugs and gene therapy vectors for DNA templates, avoiding reduced efficacy and increased immunogenicity caused by erroneous transcription / translation.

[0165] 2. Production increased significantly (2.5-3 times) Yield of this invention's method: 12.5 μg / 100 μL (1 kb fragment); Yield of conventional PCR: 4.5 μg / 100 μL Increased by 2.78 times; Technological breakthrough: By separating and replacing the substrate with magnetic beads in the middle stage, the dNTP concentration was restored from 50-80 μM to 200 μM, and the pyrophosphate concentration was reduced from 1-2 mM to <0.01 mM, "restarting" the PCR reaction, extending the efficient amplification period, and breaking through the plateau limitation.

[0166] Extremely high purity

[0167] Technological breakthrough: Magnetic beads selectively bind to target-sized DNA (>200 bp), removing short fragments, primers, proteins, and small molecule inhibitors, achieving purity close to that of commercial DNA purification kits (but with higher recovery rates, >90% vs 70-85%).

[0168] Application value: No additional purification steps are required; it can be used directly for downstream applications. Reduced dsRNA contamination in IVT (from 2.3% to <0.1%). Improve sequencing accuracy and cloning efficiency.

[0169] Excellent mRNA transcription performance

[0170] Technological breakthrough: For the first time, it has been demonstrated that DNA templates prepared by PCR can achieve IVT effects comparable to plasmids (mRNA yield is only 7.6% lower, with no statistically significant difference), but the preparation time is shortened by 87% (4.5h vs 36h) and the cost is reduced by 81%.

[0171] Application value: It accelerates the development cycle of mRNA drugs (eliminating the need for plasmid construction and bacterial culture); enables rapid response (such as rapid production of mutant vaccines during epidemics); reduces GMP production costs (plasmids require large-scale bacterial fermentation, while this invention only requires in vitro enzymatic reactions); and allows for flexible introduction of modifications (such as pseudouridine and N1-methylpseudouridine).

[0172] 5. High process stability, suitable for industrialization. Inter-batch variation CV: <10% (GMP requirement <15%); Intra-batch variation CV: <11%; Pass rate: >96% Scalable to 96-well / 384-well high-throughput automation; Technological breakthrough: Achieves highly repeatable processes through explicit CPP (Critical Process Parameters) and CQA (Critical Quality Attributes) control. The magnetic bead separation step is easily automated, overcoming the bottleneck of traditional column purification's difficulty in high-throughput processing.

[0173] Reduced manual operation time by 90% (from 43.2h to 45min, 96 samples).

[0174] 7. Easy to operate and takes a moderate amount of time. Total time: 4.5 hours (traditional PCR 3 hours, but requires 3 replicates = 9 hours); Magnetic bead separation: 30 minutes (low technical threshold); Easy to learn: follow SOP, success rate >95%; 8. Wide range of applications Fragment length: 200 bp to 10 kb (optimal 500 bp-5 kb); Template type: genomic DNA, plasmid, cDNA, synthetic DNA; Polymerase: suitable for various high-fidelity enzymes such as Phusion, Q5, PrimeSTAR, and Pfu; Application areas: mRNA IVT, NGS library construction, CRISPR gRNA template, gene synthesis, cloning, etc. 9. Environmentally friendly Ethanol dosage: 400 μL / reaction (recoverable); no toxic organic solvents (such as phenol, chloroform); magnetic beads can be reused multiple times (after recycling).

[0175] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are only used to illustrate the technical solutions of the present invention, not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A DNA amplification method, characterized in that, The method includes: S1. Perform N1 PCR cycles in a first PCR reaction solution containing at least template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme to obtain the first stage solution; S2. Contact the first-stage solution with the target solid-phase support to bind the target solid-phase support with the target DNA product to form a carrier conjugate; separate the carrier conjugate from the first-stage solution; S3. The vector conjugate is eluted in a second PCR reaction solution containing at least primers, dNTPs, DNA polymerase and mismatch recognition enzyme to release the target DNA product. The target solid-phase vector after releasing the target DNA product is separated from the second PCR reaction solution to obtain a second-stage solution. S4. Perform N2 PCR cycles in the second stage solution to obtain an amplification solution containing the target DNA product; Wherein, N1 is a preset first cycle number, and N2 is a preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, CEL I nuclease, EndoMS enzyme, and Surveyor enzyme.

2. The DNA amplification method according to claim 1, characterized in that, The mismatch recognition enzyme is T7 endonuclease I; wherein: the concentration of T7 endonuclease I in the first PCR reaction solution is 0.01-0.1 U / μL; and the concentration of T7 endonuclease I in the second stage solution is 0.005-0.05 U / μL.

3. The DNA amplification method according to claim 2, characterized in that, The concentration of T7 endonuclease I in the first PCR reaction solution is 0.025-0.075 U / μL; the concentration of T7 endonuclease I in the second stage solution is 0.0125-0.0375 U / μL.

4. The DNA amplification method according to claim 1, characterized in that, The DNA polymerase has 3'→5' exonuclease activity, and the DNA polymerase is at least one of Phusion DNA polymerase, Q5 DNA polymerase, PrimeSTAR DNA polymerase, and Pfu DNA polymerase.

5. The DNA amplification method according to any one of claims 1-4, characterized in that, The N1 is set to 10-25, and the N2 is set to 10-25.

6. The DNA amplification method according to claim 5, characterized in that, The N1 is set to 16-19, the N2 is set to 13-18, and the N1+N2 is set to 30-37.

7. The DNA amplification method according to claim 1, characterized in that, The target solid support is hydroxyl-modified magnetic beads, and step S2 specifically includes: The first-stage solution is mixed with the target solid-phase support in a PEG-salt solution to bind the target solid-phase support with the target DNA product to form a carrier conjugate. The carrier conjugate was obtained by magnetic separation and then washed with ethanol.

8. The DNA amplification method according to claim 7, characterized in that, The PEG-salt solution consists of 15%-22% w / v polyethylene glycol and 2.0-3.0 M NaCl.

9. The DNA amplification method according to claim 8, characterized in that, The second PCR reaction solution includes at least the following components: dNTPs: 150-400 μM dATP, 150-400 μM dGTP, 150-400 μM dTTP and 150-400 μM dCTP; Primers: 0.1-0.5 μM forward primer and 0.1-0.5 μM reverse primer; DNA polymerase at a concentration of 0.005-0.02 U / μL; 0.005-0.05 U / μL of mismatch recognition enzyme; 1.0-2.0 mM Mg² + .

10. The DNA amplification method according to claim 9, characterized in that, The second PCR reaction solution includes at least the following components: dNTPs: 200-300 μM dATP, 200-300 μM dGTP, 200-300 μM dTTP and 200-300 μM dCTP; Primers: 0.2-0.3 μM forward primer and 0.2-0.3 μM reverse primer; 0.01 U / μL DNA polymerase; 0.005-0.05 U / μL of mismatch recognition enzyme; 1.0-2.0 mM Mg² + .

11. A DNA amplification device, characterized in that, The device includes: A first reaction chamber, comprising at least a first PCR reaction solution containing template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme; the first reaction chamber is used to control the first PCR reaction solution to undergo N1 PCR cycles to obtain a first-stage solution. A purification device is connected to the first reaction chamber. The first-stage solution is brought into contact with the target solid-phase support in the purification device to bind the target solid-phase support with the target DNA product to form a carrier conjugate. The purification device is used to separate the carrier conjugate from the first-stage solution. The second reaction chamber contains at least a second PCR reaction solution comprising primers, dNTPs, DNA polymerase, and mismatch recognition enzyme. The second reaction chamber is used to elute the vector conjugate in the second PCR reaction solution to release the target DNA product. The second reaction chamber is also used to separate the target solid-phase vector after the release of the target DNA product from the second PCR reaction solution to obtain a second-stage solution. The second-stage solution undergoes N2 PCR cycles in the second reaction chamber to obtain an amplification solution containing the target DNA product; Wherein, N1 is a preset first cycle number, and N2 is a preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, and CEL I nuclease.

12. A DNA amplification system, characterized in that, The system includes: A liquid processing workstation is used to prepare a first PCR reaction solution containing at least template DNA, primers, dNTPs, DNA polymerase, and mismatch recognition enzyme, and then dispense the first PCR reaction solution into a first 96-well PCR plate. The first 96-well PCR plate is used to contain the first PCR reaction solution, so that N1 PCR cycles are performed in the first PCR reaction solution to obtain the first stage solution; A deep-well plate is used to contain the first-stage solution, and the deep-well plate is also configured with target magnetic beads so that the target magnetic beads bind to the target DNA product to form a carrier conjugate. A 96-well magnetic rack is used to separate the carrier conjugate from the first-stage solution; The deep well plate is also used to add a second PCR reaction solution containing at least primers, dNTPs, DNA polymerase and mismatch recognition enzyme, and to elute the vector conjugate in the second PCR reaction solution to release the target DNA product. The target solid-phase vector after releasing the target DNA product is separated from the second PCR reaction solution to obtain a second-stage solution. The second 96-well PCR plate is used to contain the second stage solution, so that N2 PCR cycles are performed in the second stage solution to obtain an amplification solution containing the target DNA product. Wherein, N1 is a preset first cycle number, and N2 is a preset second cycle number; the mismatch recognition enzyme is at least one of T7 endonuclease I, MutS protein, and CEL I nuclease.

13. A method for preparing mRNA, characterized in that, The method includes: The target DNA template is prepared by the DNA amplification method according to any one of claims 1-10, wherein the target DNA template includes a target gene coding sequence CDS, and further includes one of the T7 RNA polymerase promoter sequence, the T3 RNA polymerase promoter sequence, and the SP6 RNA polymerase promoter sequence; The target DNA template was used to perform an in vitro transcription reaction to obtain the target mRNA.

14. The method for preparing mRNA according to claim 13, characterized in that, The process of using the target DNA template to perform an in vitro transcription reaction to obtain the target mRNA includes: During the in vitro transcription reaction using the target DNA template, a Cap0 or Cap1 structure is introduced at the 5' end of the mRNA using co-transcriptional capping or post-transcriptional capping. A poly(A) tail of 50-250 nucleotides is then added to the 3' end of the mRNA using Poly(A) polymerase to obtain the target mRNA.

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