A high-fidelity gene synthesis method based on column synthesis and three-step integrated assembly

Abstract

The application provides a high-fidelity gene synthesis method based on column synthesis and three-step linkage integrated assembly, and belongs to the technical field of genetic engineering. The method comprises the following steps: using a computer program to equally divide a target gene into oligonucleotide fragments with a length of 150 nt-180 nt, and designing orthogonal 4 bp sticky ends and universal primer binding sites for each fragment; after synthesizing the above-mentioned fragments by using a column DNA synthesizer, performing equimolar mixing, and taking the mixed fragments as a template to perform single-tube PCR synchronous amplification; sequentially performing three-step linkage processes of Golden Gate Assembly directional pre-connection, full-length specific PCR enrichment amplification and Gibson Assembly seamless final assembly on the amplification products, and finally obtaining a complete target gene. The application integrates the low error rate advantage of column synthesis and the multi-step collaborative assembly strategy, significantly improves the synthesis fidelity and success rate, is especially suitable for rapid and low-cost preparation of complex genes with a GC content of 30%-60% and containing repetitive sequences, and has the characteristics of simple operation, strong device versatility, suitability for large-scale application and the like.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a high-fidelity gene synthesis method based on column synthesis and three-step integrated assembly. Background Technology

[0002] Gene synthesis, or de novo construction of target DNA sequences, is a core enabling technology in molecular biology, synthetic biology, and related biotechnology fields. It provides fundamental materials for applications such as gene function research, protein engineering, metabolic pathway reconstruction, gene therapy vector development, and DNA information storage. For genes exceeding the chemical synthesis limit of single-stranded oligonucleotides (typically >200-300 nt), the industry generally adopts a "segmented synthesis-assembly" strategy: first, the target gene is broken down into shorter oligonucleotide fragments for chemical synthesis, and then these fragments are accurately assembled into a complete long-chain gene using enzymatic methods.

[0003] Currently, the chemical synthesis of oligonucleotides mainly relies on solid-phase synthesis techniques, which can be broadly divided into two categories: high-throughput chip synthesis and column synthesis. Chip synthesis allows for the parallel synthesis of thousands to tens of thousands of different oligonucleotides on a single chip, offering significant throughput advantages. However, its single-base error rate is typically high (approximately 0.1%), and the yield per sequence is extremely low (fmol level), requiring independent PCR amplification to meet the quantity needed for downstream assembly. This amplification step not only increases operation time and cost but may also introduce additional mutations. In contrast, while column synthesis has a limited sequence throughput per synthesis (typically ≤384 sequences), it can achieve a lower inherent error rate (single-base error rate ≤0.05%) and a single-column yield in the nmol to μmol level by optimizing the solid-phase support (such as CPG) and synthesis process. High-purity column-synthesized products can usually be used directly for subsequent assembly without additional amplification, demonstrating advantages in terms of synthesis fidelity and product usability.

[0004] After obtaining oligonucleotide fragments, efficient and accurate assembly remains another key technological challenge. In existing technologies, computer-aided design has been applied to this process; for example, the OMEGA assembly method and its accompanying DAD differential algorithm can automatically segment long genes into fragments and design 4 bp orthogonal sticky ends for Golden Gate assembly, thereby achieving directional ligation of multiple fragments. However, such methods are often deeply coupled with chip synthesis technologies with high error rates and lack integrated effective error correction or quality control units. When dealing with genes with abnormal GC content or complex repetitive structures, assembly accuracy and success rates can be affected.

[0005] Currently used genome assembly methods each have their limitations. Golden Gate assembly technology relies on type IIs restriction endonucleases, and its efficiency decreases sharply with the increase in the number of fragments assembled (e.g., more than 10), easily leading to non-specific ligation and chimeras. Methods such as Gibson Assembly are highly dependent on precise homologous sequence pairing at the fragment ends, exhibiting poor compatibility with templates with complex secondary structures or repetitive sequences. Overlap extension PCR (SOE-PCR) has low amplification efficiency and poor specificity for long fragments (>2 kb) or complex templates, with a success rate often below 60%. All these methods struggle to efficiently and stably assemble challenging complex gene sequences while maintaining high fidelity.

[0006] Furthermore, for column-based synthesis, overcoming the limitations of single-batch synthesis throughput and establishing a standardized, low-cost process capable of stably synthesizing "difficult" genes such as those with long lengths (e.g., 800 bp), abnormal GC content (e.g., 30%-60% overall), or containing tandem repeat sequences remains a pressing technical challenge in this field. Existing technical solutions generally suffer from drawbacks such as cumbersome processes, long processing times, large fluctuations in success rates, and high overall costs, making it difficult to meet the demands of research and industry for rapid, high-fidelity, and scalable gene synthesis. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a high-fidelity gene synthesis method based on column synthesis and three-step linkage integrated assembly, so as to overcome the defects of the prior art, such as high error rate of chip synthesis, poor compatibility of assembly process with complex genes, and cumbersome and inefficient multi-tube operation.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a high-fidelity gene synthesis method based on column synthesis and three-step integrated assembly, the method comprising the following steps:

[0010] (a) The nucleotide sequence of the target gene is automatically segmented and the ends are designed by a computer program to output N oligonucleotide fragment sequences, wherein N≥2, the length L of each fragment satisfies 150 nt≤L≤180 nt, and orthogonal 4 bp sticky ends are designed at both ends of each fragment, which are generated after being cleaved by Type IIs restriction endonucleases;

[0011] (b) Synthesize the oligonucleotide fragment described in step (a) using a column DNA synthesizer;

[0012] (c) Mix the oligonucleotide fragments synthesized in step (b) in equal molar amounts, and use the mixture as a single template to perform a single-tube PCR amplification reaction using a pair of universal primers;

[0013] (d) The double-stranded DNA fragment product obtained in step (c) is assembled using a three-step assembly process: preliminary assembly is performed using Golden Gate Assembly, in a reaction system containing Type II restriction endonucleases and DNA ligases, the fragments are directionally ligated through the 4 bp sticky ends; using the preliminary assembly product as a template, PCR amplification is performed using specific primers at both ends corresponding to the full-length sequence of the target gene; the PCR amplification product is then finally assembled using Gibson Assembly to obtain the full-length target gene.

[0014] Preferably, in step (a), the operation performed by the computer program includes: splitting all oligonucleotide fragments to the exact same length.

[0015] Preferably, the length is made identical by adding non-functional random filler sequences to all oligonucleotide fragments.

[0016] Preferably, in step (a), universal primer binding sites at the 5' and 3' ends are uniformly added to all oligonucleotide fragments; the pair of universal primers in step (c) are complementary to the universal primer binding sites.

[0017] Preferably, in step (a), the computer program employs the DAD algorithm.

[0018] Preferably, the overall GC content of the target gene is between 30% and 60%.

[0019] Preferably, the target gene contains a tandem repeat sequence of length ≥15 bp.

[0020] Secondly, the present invention provides the application of the method described in the first aspect in the synthesis of complex genes, wherein the complex gene is a gene with a length not exceeding 1000bp, an overall GC content between 30% and 60%, and contains tandem repeat sequences.

[0021] The beneficial effects of this invention are:

[0022] 1. By employing an integrated strategy of "Golden Gate initial assembly—PCR targeted enrichment—Gibson final assembly," the limitations of single assembly technologies for complex sequences (such as those with high / low GC content or repetitive sequences) are effectively overcome. In an example involving a complex gene of 800 bp, 12 fragments were used for assembly, and sequencing verification showed an accuracy rate of 67%, effectively improving the success rate and stability of complex gene synthesis.

[0023] 2. By pre-designing universal primer binding sites for all synthesized fragments, simultaneous PCR amplification in a single tube is achieved after mixing all fragments in equal molar amounts, completely eliminating the cumbersome traditional method of independently aliquoting and amplifying each fragment. This innovation simplifies more than 70% of manual operation steps and saves more than 50% of PCR-related reagents and consumables.

[0024] 3. Utilizing column synthesis technology with a lower error rate from the synthesis source reduces the introduction of initial errors. The subsequent three-step linkage assembly strategy effectively eliminates incorrectly connected or unconnected short fragments, further ensuring the sequence accuracy of the final product.

[0025] 4. The entire design process is fully automated, relying on computer programs to automatically complete gene segmentation of equal length, design of orthogonal sticky ends, addition of universal adapters, and length uniformity through filler sequences. This automated design process eliminates errors that may be introduced by manual operation, ensuring high reproducibility and standardization of the scheme, and is suitable for batch synthesis.

[0026] 5. The entire synthesis and assembly process only requires a commercially available column-type DNA synthesizer and a regular PCR instrument, making the equipment highly versatile and easy to promote and implement. Attached Figure Description

[0027] Figure 1 The image shows the agarose gel electrophoresis results of the globally mixed high-purity double-stranded fragment product obtained by medium-molar homogeneous mixing and amplification in Example 1.

[0028] Figure 2 The image shows the agarose gel electrophoresis result of the 800bp product obtained by PCR enrichment and amplification in Example 1.

[0029] Figure 3 The image shows PCR gel images of eight randomly selected monoclonal colonies from Example 1. Detailed Implementation

[0030] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0031] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0032] Example 1: Synthesis of L0-14-2N gene (sequence complexity level: Complex)

[0033] 1. Gene sequence

[0034] The target gene is a complex gene with a length of 800 bp, and its nucleotide sequence is shown in SEQ ID NO: 15. Its overall GC content is about 50%, and it contains tandem repeat sequences.

[0035] 2. Computer-aided design and synthesis of oligonucleotide fragments

[0036] A computer program based on the DAD algorithm is used to automatically design the above target gene sequence. The program performs the following operations:

[0037] (a) The full-length 800 bp gene was equally divided into 12 coding oligonucleotide fragments, each with a target length of 150 nt.

[0038] (b) Design 4 bp sticky ends that can be generated by BsaI (Type IIs) restriction endonucleases at both ends of each fragment to ensure directionality and specificity during assembly.

[0039] (c) A fixed universal forward primer binding sequence (SEQ ID NO:13) was uniformly added to the 5' end of all 12 fragments, and a fixed universal reverse primer binding sequence (SEQ ID NO:14) was uniformly added to the 3' end.

[0040] (d) By inserting non-biologically active random filler sequences into some fragments, the final synthetic lengths of all 12 fragments were calibrated to be completely consistent, with the length deviation controlled within 1 nt.

[0041] (e) The program outputs 12 oligonucleotide synthesis sequences suitable for column DNA synthesizers (specific sequences are shown in SEQ ID NO:1 to SEQ ID NO:12) and a pair of corresponding universal amplification primer sequences (forward: SEQ ID NO:13; reverse: SEQ ID NO:14) with one click.

[0042] Subsequently, the above 12 oligonucleotide fragments were synthesized using a column-based DNA synthesizer. After synthesis, the fragments were deprotected and purified to obtain high-purity single-stranded DNA products.

[0043] 3. Equimolar mixing of fragments and simultaneous single-tube PCR amplification

[0044] The molar concentration of each purified fragment was determined and calculated using a nucleic acid quantification instrument. The 12 fragments were then mixed strictly in equimolar proportions to prepare a single equimolar mixed template solution.

[0045] Take 1 μL of the above mixed template and place it in a 20 μL PCR reaction system for single-tube simultaneous amplification. The reaction system contains: 0.5 μM each of universal forward primer (SEQ ID NO:13) and universal reverse primer (SEQ ID NO:14), 17 μL of 1.25× commercial PCR premix, and bring the volume to 20 μL with sterile enzyme-free water. The PCR reaction program is as follows: 95℃ pre-denaturation for 2 minutes; then 16 cycles: 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 30 seconds; and finally 72℃ final extension for 2 minutes. The amplified products are verified by agarose gel electrophoresis (e.g., ...). Figure 1 The bands showed a single, clear band of approximately 150 bp, indicating that all fragments were amplified uniformly and specifically.

[0046] 4. Three-step integrated assembly

[0047] Step 1: Initial assembly of the Golden Gate.

[0048] Take approximately 100 ng of the single-tube PCR amplification product obtained in step 3, add 1.5 μL of BsaI (Type IIs) restriction endonuclease, 1.5 μL of T4 DNA ligase, and 2 μL of dedicated 10×Golden Gate reaction buffer, and bring the volume to 20 μL with sterile water. Perform the reaction using the following thermal cycling program: incubate at 37°C for 1 minute, then perform 25 cycles (37°C for 5 minutes, 16°C for 5 minutes), followed by incubation at 50°C for 15 minutes, and finally incubation at 80°C for 15 minutes to inactivate the enzyme. This step utilizes pre-designed orthogonal 4 bp sticky ends to achieve directional pre-ligation of the fragment.

[0049] Step 2: Full-length specific PCR targeted enrichment and amplification.

[0050] Using 1 μL of the initial Golden Gate assembly reaction solution from step one as a template, high-fidelity PCR amplification was performed using specific primers designed for the 5' and 3' ends of the target gene's full-length sequence. The PCR extension time was set to 2 minutes to ensure complete amplification of the 800 bp full-length product. After amplification, the approximately 800 bp target band of the correct size (e.g., [image of target gene]) was recovered by agarose gel electrophoresis. Figure 2 ).

[0051] Step 3: Gibson seamless closed-loop final assembly.

[0052] Take approximately 50 ng of the PCR purified product recovered in step two and mix it with 20 μL of commercially available Gibson Assembly MasterMix. Make up the total reaction volume to 40 μL with sterile water. Incubate the reaction mixture at 50°C for 60 minutes. This premix contains T5 exonuclease, DNA polymerase, and thermostable DNA ligase, which can repair any terminal single-strand gaps in the pre-ligated product, achieving seamless, closed-loop assembly of the full-length gene.

[0053] 5. Cloning and Verification

[0054] 5 μL of the Gibson final assembly product obtained in step 3 was transformed into DH5α E. coli competent cells, plated on LB agar containing ampicillin, and incubated overnight. Eight single clones were randomly selected for colony PCR identification (e.g., ...). Figure 3 The positive rate was 100%. Three positive clones were randomly selected for full-length gene sequencing analysis. The sequencing results showed that the sequences of two clones were completely identical to the target sequence, with no base mutations, insertions, or deletions, and the assembly accuracy was 67%. This result confirms the high success rate of this method in synthesizing complex genes.

[0055] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A high-fidelity gene synthesis method based on column synthesis and three-step integrated assembly, characterized in that, Includes the following steps: (a) The nucleotide sequence of the target gene is automatically segmented and the ends are designed by a computer program, and N oligonucleotide fragment sequences are output, wherein N≥2, the length L of each fragment satisfies 150 nt≤L≤180 nt, and orthogonal 4 bp sticky ends are designed at both ends of each fragment, which are generated after being cleaved by Type II restriction endonucleases; (b) Synthesize the oligonucleotide fragment described in step (a) using a column DNA synthesizer; (c) Mix the oligonucleotide fragments synthesized in step (b) in equal molar amounts, and use the mixture as a single template to perform a single-tube PCR amplification reaction using a pair of universal primers; (d) The double-stranded DNA fragment product obtained in step (c) is assembled using a three-step assembly process: GoldenGate Assembly is performed for preliminary assembly, in a reaction system containing Type IIs restriction endonuclease and DNA ligase, the fragments are directionally ligated through the 4 bp sticky ends; using the preliminary assembly product as a template, PCR amplification is performed using specific primers at both ends corresponding to the full-length sequence of the target gene; the PCR amplification product is then finally assembled using Gibson Assembly to obtain the full-length target gene.

2. The method according to claim 1, characterized in that, In step (a), the computer program performs the following operations: splitting all oligonucleotide fragments to the exact same length.

3. The method according to claim 2, characterized in that, The lengths were made identical by adding non-functional random filler sequences to all oligonucleotide fragments.

4. The method according to claim 1, characterized in that, In step (a), universal primer binding sites are uniformly added to the 5' and 3' ends of all oligonucleotide fragments; the pair of universal primers mentioned in step (c) are complementary to the universal primer binding sites.

5. The method according to claim 1, characterized in that, In step (a), the computer program employs the DAD algorithm.

6. The method according to claim 1, characterized in that, The overall GC content of the target gene is between 30% and 60%.

7. The method according to claim 1, characterized in that, The target gene contains tandem repeat sequences of ≥15 bp in length.

8. The application of the method according to any one of claims 1-7 in the synthesis of complex genes, characterized in that, The complex gene is a gene with a length not exceeding 1000bp, an overall GC content between 30% and 60%, and containing tandem repeat sequences.

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