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Synthetic Biology

Volume 1073 of the series Methods in Molecular Biology pp 9-17

Date:

Gene Synthesis Method Based on Overlap Extension PCR and DNAWorks Program

  • Gang LiAffiliated withState Key Laboratory of Biocontrol, Sun Yat-sen University
  • , Bing-Xue DongAffiliated withState Key Laboratory of Biocontrol, Sun Yat-sen University
  • , Yu-Huan LiuAffiliated withState Key Laboratory of Biocontrol, Sun Yat-sen University
  • , Chang-Jie LiAffiliated withState Key Laboratory of Biocontrol, Sun Yat-sen University
  • , Li-Ping ZhangAffiliated withState Key Laboratory of Biocontrol, Sun Yat-sen University

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Abstract

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