[An efficient assembly method for a viral genome based on T7 endonuclease Ⅰ-mediated error correction]

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genomics

Context:

• Current de novo gene synthesis methods face challenges with tedious operations, low efficiency, high error rates, and limited product lengths.
• These limitations hinder the large-scale demands of synthetic biology.
• Efficient gene synthesis is crucial for advancing fields like synthetic biology.

Purpose:

• To develop an efficient de novo gene synthesis method for a 10 kb viral genome.
• To optimize oligonucleotide design, PCR amplification, and error correction for large DNA fragment synthesis.
• To reduce error rates and simplify the gene synthesis and assembly process.

Summary:

• Designed oligonucleotide sequences for a 10 kb virus genome, balancing software, PCR, and assembly enzyme capabilities.
• Performed two-step PCR with high-fidelity polymerase and T7 endonuclease I error correction to synthesize 3.0 kb DNA fragments.
• Assembled and sequenced the 10 kb viral genome from synthesized fragments, achieving an error rate as low as 0.36 errors/kb.

Impact:

• Successfully synthesized a 10 kb viral genome in one day and its correct plasmid in five days.
• Significantly reduced the probability of large fragment mutations during assembly.
• Optimized de novo gene synthesis, lowering error rates, simplifying steps, and decreasing costs for viral genome assembly.