title: New insights into the de novo gene synthesis using the automatic kinetics switch approach.
abstract: Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6kbp from 1nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74kbp from 1nM oligonucleotide.
authors.affiliation: Institute of Bioengineering and Nanotechnology, The Nanos, Singapore, Singapore.
authors.forename: Wai Chye
authors.initials: WC
authors.lastname: Cheong
authors.forename: Li Shi
authors.initials: LS
authors.lastname: Lim
authors.forename: Mo Chao
authors.initials: MC
authors.lastname: Huang
authors.forename: Marcus
authors.initials: M
authors.lastname: Bode
authors.forename: Mo-Huang
authors.initials: MH
authors.lastname: Li
mesh_terms: D001331:Automation; D002021:Buffers; D017931:DNA Primers / Q000235:genetics; D005796:Genes / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
chemical_list: D002021:Buffers; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides