Abstract
This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer primers is designed to be ∼8°C lower than that of the assembly oligonucleotides, and (2) different annealing temperatures are utilized to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. This method eliminates the interference between polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. Additionally, the TopDown gene synthesis has been combined with the LCGreen I DNA fluorescence dye in a real-time gene synthesis approach for investigating the stepwise efficiency and kinetics of PCR-based gene synthesis. The obtained real-time fluorescence signals are compared with gel electrophoresis results to optimize gene synthesis conditions.
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Huang, M.C., Cheong, W.C., Ye, H., Li, MH. (2012). TopDown Real-Time Gene Synthesis. In: Peccoud, J. (eds) Gene Synthesis. Methods in Molecular Biology, vol 852. Humana Press. https://doi.org/10.1007/978-1-61779-564-0_3
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DOI: https://doi.org/10.1007/978-1-61779-564-0_3
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