Presentation Information
[P04-507]Replication cycle reaction–mediated multi-plasmid synthesis from an oligo pool
○Satoru Sumi1, Masayuki Su’etsugu1 (1. Rikkyo university (Japan))
Keywords:
Gene synthesis,Cell free technology,Error removal
Recent advances in synthetic biology have increased the demand for artificial gene synthesis. In gene synthesis, oligonucleotides are used as starting materials for PCR-based assembly to generate double-stranded DNA. Oligo pools, which can contain up to one million different sequences, are attractive for reducing the cost of high-throughput gene synthesis. However, they present technical challenges, including limited quantities and sequence errors.
Here, we developed a method termed CADO (Circular Amplification Directly from Oligo assembly), which enables parallel synthesis of multiple plasmids from a low-cost, low-concentration oligo pool. In this approach, several tens of oligonucleotides are assembled by hybridization to form circular DNA containing a vector sequence, which is subsequently amplified using the replication cycle reaction (RCR). RCR is a reconstituted Escherichia coli chromosomal replication system that can amplify circular DNA even from a single molecule. Using CADO with an oligo pool containing 64 distinct oligonucleotides, we successfully constructed four different plasmids simultaneously in a single reaction, each encoding a 0.8 kb synthetic gene. We also developed an error-removal system to eliminate oligonucleotide-derived errors, thereby improving sequence accuracy. Thus, this method enables high-fidelity, multiplexed synthesis of artificial genes directly from oligo pools.
Here, we developed a method termed CADO (Circular Amplification Directly from Oligo assembly), which enables parallel synthesis of multiple plasmids from a low-cost, low-concentration oligo pool. In this approach, several tens of oligonucleotides are assembled by hybridization to form circular DNA containing a vector sequence, which is subsequently amplified using the replication cycle reaction (RCR). RCR is a reconstituted Escherichia coli chromosomal replication system that can amplify circular DNA even from a single molecule. Using CADO with an oligo pool containing 64 distinct oligonucleotides, we successfully constructed four different plasmids simultaneously in a single reaction, each encoding a 0.8 kb synthetic gene. We also developed an error-removal system to eliminate oligonucleotide-derived errors, thereby improving sequence accuracy. Thus, this method enables high-fidelity, multiplexed synthesis of artificial genes directly from oligo pools.
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