Presentation Information
[3GteX-02]Multiplex gene synthesis with error removal from oligo pools using replication cycle reaction–based circular DNA amplification
○Masayuki Su'etsugu1, Sana Shimizu1, Satoru Sumi1 (1. Rikkyo university (Japan))
Keywords:
DNA synthesis
[Purpose]
The demand for synthetic genes is increasing with advances in biotechnology; however, conventional PCR-based assembly from chemically synthesized oligonucleotides remains costly and limited in throughput. Although high-complexity oligo pools offer a promising solution, their practical use is hindered by the extremely low abundance and high error rates of individual oligos. This study aims to develop a method for efficient and accurate gene synthesis from oligo pools under cell-free conditions.
[Method]
We employed the Replication Cycle Reaction (RCR), a cell-free reconstitution of the Escherichia coli chromosomal replication system, which enables exponential amplification of circular DNA molecules. Oligo pools were first assembled into gene-length DNA fragments, which were subsequently assembled with linear vector DNA to generate circular constructs, followed by amplification via RCR. In addition, an error-removal strategy was implemented to eliminate sequence errors introduced during oligo synthesis.
[Results]
Using an ultra-low-concentration oligo pool containing 16 distinct sequences, we successfully assembled and amplified a functional sfGFP expression plasmid. Furthermore, from an oligo pool comprising 64 sequences, we achieved multiplex synthesis of four distinct fluorescent protein expression plasmids in a single tube. Incorporation of an error-removal strategy significantly improved the fidelity of the resulting plasmids.
[Consideration]
These results demonstrate that RCR enables selective and exponential amplification of correctly assembled circular DNA molecules, overcoming the limitations associated with low-abundance oligo pools. The integration of error correction further enhances sequence accuracy, addressing a major bottleneck in oligo-based gene synthesis.
[Conclusion]
We present a unique cell-free platform for multiplex plasmid synthesis from low-abundance, error-prone oligo pools. This approach provides a scalable and cost-effective strategy for high-throughput gene synthesis and synthetic biology applications.
The demand for synthetic genes is increasing with advances in biotechnology; however, conventional PCR-based assembly from chemically synthesized oligonucleotides remains costly and limited in throughput. Although high-complexity oligo pools offer a promising solution, their practical use is hindered by the extremely low abundance and high error rates of individual oligos. This study aims to develop a method for efficient and accurate gene synthesis from oligo pools under cell-free conditions.
[Method]
We employed the Replication Cycle Reaction (RCR), a cell-free reconstitution of the Escherichia coli chromosomal replication system, which enables exponential amplification of circular DNA molecules. Oligo pools were first assembled into gene-length DNA fragments, which were subsequently assembled with linear vector DNA to generate circular constructs, followed by amplification via RCR. In addition, an error-removal strategy was implemented to eliminate sequence errors introduced during oligo synthesis.
[Results]
Using an ultra-low-concentration oligo pool containing 16 distinct sequences, we successfully assembled and amplified a functional sfGFP expression plasmid. Furthermore, from an oligo pool comprising 64 sequences, we achieved multiplex synthesis of four distinct fluorescent protein expression plasmids in a single tube. Incorporation of an error-removal strategy significantly improved the fidelity of the resulting plasmids.
[Consideration]
These results demonstrate that RCR enables selective and exponential amplification of correctly assembled circular DNA molecules, overcoming the limitations associated with low-abundance oligo pools. The integration of error correction further enhances sequence accuracy, addressing a major bottleneck in oligo-based gene synthesis.
[Conclusion]
We present a unique cell-free platform for multiplex plasmid synthesis from low-abundance, error-prone oligo pools. This approach provides a scalable and cost-effective strategy for high-throughput gene synthesis and synthetic biology applications.
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