A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

PloS One
Jiang-Ke YangJiang-Hong Dai

Abstract

In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

References

May 10, 2002·Nucleic Acids Research·David M Hoover, Jacek Lubkowski
Dec 6, 2003·Proceedings of the National Academy of Sciences of the United States of America·Hamilton O SmithJ Craig Venter
Apr 17, 2004·Nucleic Acids Research·Lei Young, Qihan Dong
Jun 25, 2004·Nucleic Acids Research·Jean-Marie RouillardErdogan Gulari
May 25, 2005·Nucleic Acids Research·Sebastian JayarajDaniel V Santi
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Apr 26, 2011·Nature Biotechnology·Jiayuan QuanJingdong Tian

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Datasets Mentioned

BETA
GQ502721
JN252710

Methods Mentioned

BETA
chip
PCR

Software Mentioned

GeMS
DNAWorks
Gene2Oligo
RNAfold

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