Gene Synthesis Method Based on Overlap Extension PCR and DNAWorks Program

  • Gang Li
  • Bing-Xue Dong
  • Yu-Huan Liu
  • Chang-Jie Li
  • Li-Ping Zhang
Part of the Methods in Molecular Biology book series (MIMB, volume 1073)


Gene synthesis by chemical methods provides a powerful tool for modifying genes and exploring their structure, expression, and function in the post-genomic era. However, a bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. Here, we describe a simple, rapid, and low-cost gene synthesis method based on overlap extension PCR (OE-PCR) and the DNAWorks program. This method enables DNA sequences with sizes ranging from 200 bp to 3 kb to be synthesized with few errors, and these errors can be easily corrected by site-directed mutagenesis. Thus, it is amenable to automation for the multiplexed synthesis of different genes and has a potential for high-throughput gene synthesis.

Key words

Gene synthesis DNAWorks Overlap extension PCR Site-directed mutagenesis 


  1. 1.
    Van Hest JCM, Tirrell DA (2001) Protein-based material, toward a new level of structural control. Chem Commun 19:1897–1904CrossRefGoogle Scholar
  2. 2.
    Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353CrossRefGoogle Scholar
  3. 3.
    Xiong AS, Yao QH et al (2005) High level expression of a recombinant acid phytase gene in Pichia pastoris. J Appl Microbiol 98:418–428CrossRefGoogle Scholar
  4. 4.
    Xiong AS, Yao QH et al (2006) High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris. Appl Microbiol Biotechnol 72:1039–1047CrossRefGoogle Scholar
  5. 5.
    Yang ZY, Kong WP et al (2004) A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 428:561–564CrossRefGoogle Scholar
  6. 6.
    Scarpulla RC, Narang S, Wu R (1982) Use of a new retrieving adaptor in the cloning of a synthetic human insulin A-chain gene. Anal Biochem 121:356–365CrossRefGoogle Scholar
  7. 7.
    Mandecki W, Bolling TJ (1988) FokI method of gene synthesis. Gene 68:101–107CrossRefGoogle Scholar
  8. 8.
    Dillon PJ, Rosen CA (1990) A rapid method for the construction of synthetic genes using the polymerase chain reaction. Biotechniques 9:298–300Google Scholar
  9. 9.
    Chen GQ, Choi I, Ramachandran B, Gouaux JE (1994) Total gene synthesis: novel single step and convergent strategies applied to the construction of a 779 base pair bacteriorhodopsin gene. J Am Chem Soc 116:8799–8800CrossRefGoogle Scholar
  10. 10.
    Hayashi N, Welschof M et al (1994) Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR. Biotechniques 17:310–315Google Scholar
  11. 11.
    Sandhu GS, Aleff RA, Kline BC (1992) Dual asymmetric PCR: one-step construction of synthetic genes. Biotechniques 12:14–16Google Scholar
  12. 12.
    Stemmer WP, Crameri A et al (1995) Single step assembly of a gene and entire plasmid from large number of oligodeoxyribonucleotides. Gene 164:49–53CrossRefGoogle Scholar
  13. 13.
    Strizhov N, Keller M et al (1996) A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc Natl Acad Sci U S A 93:15012–15017CrossRefGoogle Scholar
  14. 14.
    Lin Y, Cheng X, Clark TG (2002) The use of synthetic genes for the expression of ciliate proteins in heterologous systems. Gene 288:85–94CrossRefGoogle Scholar
  15. 15.
    Gao X, Yo P et al (2003) Thermodynamically balanced inside-out (TBIO) PCR-based synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acids Res 31:e143CrossRefGoogle Scholar
  16. 16.
    Young L, Dong QH (2004) Two-step total gene synthesis method. Nucleic Acids Res 32:e59CrossRefGoogle Scholar
  17. 17.
    An Y, Ji J et al (2005) A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR. Appl Microbiol Biotechnol 68:774–778CrossRefGoogle Scholar
  18. 18.
    Dong BX, Mao RQ et al (2007) An improved method of gene synthesis based on DNAWorks software and overlap extension PCR. Mol Biotechnol 37:195–200CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Gang Li
    • 1
  • Bing-Xue Dong
    • 1
  • Yu-Huan Liu
    • 1
  • Chang-Jie Li
    • 1
  • Li-Ping Zhang
    • 1
  1. 1.State Key Laboratory of BiocontrolSun Yat-sen UniversityGuangzhouChina

Personalised recommendations