New insights into the de novo gene synthesis using the automatic kinetics switch approach.
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.
Cheong
Wai Chye
WC
Institute of Bioengineering and Nanotechnology, The Nanos, Singapore, Singapore.
Lim
Li Shi
LS
Huang
Mo Chao
MC
Bode
Marcus
M
Li
Mo-Huang
MH
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
D002021:Buffers; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides