Simplified gene synthesis: a one-step approach to PCR-based gene construction

Journal of Biotechnology
Gang WuStephen J Freeland

Abstract

PCR-based gene synthesis conventionally requires two steps: first, all overlapping oligonucleotides are assembled by self-priming; then an additional pair of primers is used to amplify the full-length gene product. Here we propose a simplified method of gene synthesis which combines these two steps into one. We have found that the efficiency of this one-step method, which we term "Simplified Gene Synthesis", is affected by multiple parameters of the PCR reactions. In particular, the choice of polymerase is critical for successful one-step assembly. Other important factors include the concentration of assembly oligonucleotides and amplification primers. Moreover, we offer a general method to estimate, given a known mutation rate, how many clones should be sequenced in order to be confident of obtaining at least one correct gene product. Having determined the accuracy of gene products synthesized under optimal conditions with Simplified Gene Synthesis, we show that our estimation works well. Overall, the simplified gene synthesis provides an easier and more efficient approach to gene synthesis, providing a further step towards the future goal of generalized automation for this process.

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Citations

May 20, 2009·Journal of the American Chemical Society·Jing XuJumi A Shin
Aug 23, 2012·The Journal of Biological Chemistry·Varsha RaoCharles J Bieberich
Oct 26, 2006·Nucleic Acids Research·Gang WuStephen J Freeland
Mar 7, 2009·Nucleic Acids Research·Hongye YeJackie Y Ying
May 1, 2008·BMC Biotechnology·Damien MarsicJoseph D Ng
Jul 17, 2008·PloS One·Jean PeccoudSally A Waldon
Feb 23, 2013·Nanomedicine : Nanotechnology, Biology, and Medicine·Peter M MoyleIstvan Toth
Sep 15, 2012·Analytical Biochemistry·Qiuting LohTheam Soon Lim
Aug 15, 2015·Journal of Microbiological Methods·Gonzalo Torres TejerizoSusana Brom
Jul 6, 2010·Analytical Biochemistry·Wai Chye CheongMo-Huang Li
Oct 6, 2009·Journal of Microbiological Methods·Joel R TerMaatAnuradha Subramanian
Jul 29, 2008·Protein Expression and Purification·Yuri Y LonderFrank R Collart
Aug 28, 2007·Journal of Biotechnology·Sandrine Denis-QuanquinDidier Fournier
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Apr 9, 2008·FEMS Microbiology Reviews·Ai-Sheng XiongQuan-Hong Yao
Jun 24, 2016·Molecular Biotechnology·Ana Filipa SequeiraCarlos M G A Fontes
Apr 4, 2017·Journal of Biotechnology·Peiching ChangClaire R Shen
Sep 21, 2007·Molecular Systems Biology·Gang WuStephen J Freeland
Jun 30, 2009·Molecular BioSystems·Jingdong TianIshtiaq Saaem
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Sep 14, 2019·Applied Microbiology and Biotechnology·Veronika V ShchurAleksei V Yantsevich
Jan 17, 2021·Biomolecules·Hyesu SeoWoong June Park
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May 10, 2013·ACS Synthetic Biology·Arun S Rajkumar, Sebastian J Maerkl

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