An Improved Method of Gene Synthesis Based on DNA Works Software and Overlap Extension PCR |
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Authors: | Bingxue Dong Runqian Mao Baojian Li Qiuyun Liu Peilin Xu Gang Li |
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Institution: | (1) The Key Laboratory of Gene Engineering of Ministry of Education, Sun Yat-sen University, Guangzhou, 510275, P.R. China;(2) Guangdong Entomological Institute, Guangzhou, 510260, P.R. China |
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Abstract: | A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing.
In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program
and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few
errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method,
which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped
region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid
and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction
conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully.
Bingxue Dong and Runqian Mao contributed equally to this work. |
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Keywords: | Gene synthesis DNAWorks OE-PCR |
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