Error Correction In DNA Synthesis By Immobilized MutS And Scalable Do Novo Gene Synthesis

The application of microchip-synthesized oligonucleotides (MCp-oligos) for gene synthesis involving millions of oligos is limited by the minal amount of each oligos, low quality and high complex of the synthetic DNA pool. Consequently, its broad application requires advances in the accuracy and scale of synthetic DNA. In this study, a low-cost, effective and high-throughput (up to492oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides.At first, a new error correction system was constructed. In this error correction method, random errors in the synthetic DNA molecules are exposed through re-annealing as mismatches. Mismatch-containing duplexes are removed by the affinity binding with immobilized MutS on cellulose column. Then, the flow-through is serves as template for PCR to produce the error-depleted DNA. This process can be iterated for increased fidelity. And to improve the binding effective of MICC, MutS from Thermus aquaticus (TaqMutS) and MutS from E. coli (EcoMutS) which have different binding affinity to various mismatched were combinatorial immobilized on cellulose column to remove the errors in DNA molecules. In this study, to construct a more effective, low-cost, easy-manipulated, and higher throughput error-removal method for wide application of MCp-oligos. Error removal is conducted directly on the MCp-oligos before assembly. With this error-correction method, we were able to improved a population of synthetic enhanced green fluorescent protein (EGFP) clones from0.93%to83.22%fluorescent, and decreased errors24.87-fold to final values of1error per2159bp.In additional, with this MutS immobilized cellulose column, four high-throughput error correction strategies corresponding to various scales were designed and evaluated. The first three stratrgies were applied for the synthesis of multi-gene construction (eg. genes, pathways or building blocks for larger DNA constructs). To simply the following gene assembly processing, the synthetic oligos pool were first devied into subpools by adding different pairs of priming site at each subpool oligos. Then error correction was performed. With the first strategy, each subpool (11-32oligos) from each fragment were parallel error correction simultaneously with corresponding MIC-Cs. With the second strategy, several subpool (38-56oligos) from each gene were parallel error correction simultaneously with corresponding MICCs. With the third strategy, all subpools of the entire oligos pool (479oligos) were error correced with only one MICC. In some case, the division of pool into subpool is not necessary, eg. construction of shuffling library with Mcp-oligos, all oligos of the entirl pool are assembled togather. The forth strategy which removes the errors of all oligos in one pool (the pool is not divided into subpools) with one MICC is applied for error correction in the construction of a shuffling library.Here, these high-throughput error correction strategies demonstrate by proceeding the synthesis of sMMO gene cluster, Epo A, B and C genes, lycopene biosynthesis genes and a RFPs gene library. With this method,21genes encoding a total of33.415kb pairs of DNA and a RFP shuffling library from870MCp-oligos were constructed. And the error frequency can be reduced from-14/kb to as low as0.56/kb. Moreover, a standard MICC error correction process can be finished in1.5hour with a cost of$0.374/MICC. Thus, this system provids a low-cost and efficient approach for large scale de novo DNA synthesis with MCq-oligos.