An improved method of gene synthesis based on DNA works software and overlap extension PCR
An improved method of gene synthesis based on DNA works software and overlap extension PCR
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DOI：
10.1007/s12033-007-0039-8
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发表时间：
2007-11-01
影响因子：
2.6
通讯作者：
Li, Gang
中科院分区：
文献类型：
--
作者：
Dong, Bingxue;Mao, Runqian;Li, Gang
目前基因合成技术的一个瓶颈是寡核苷酸合成和合成后测序的高成本。本文基于DNAWorks程序和改进的一步重叠延伸聚合酶链式反应(OE-PCR)，建立了一种简便、快速的低成本基因合成技术。这种方法可以无误差地合成任何DNA序列，然后通过定点突变技术或PCR扩增-组装方法纠正任何突变位点，从而扩增出目的基因的不同DNA片段，然后通过它们的重叠区组装成一个完整的基因。最终，通过这种新的方法获得了无误的全长DNA序列。我们的方法简单、快速、低成本，而且易于基于DNAWorks设计程序和定义的一套适合不同基因的OE-PCR反应条件而实现自动化。利用该方法成功合成了黄孢原毛平革菌(P.chrysosporium)的锰过氧化物酶基因(Manp)、杂色毛霉(Trametes Versicolor)的漆酶基因(Lac)和灰鸡腿霉(Coprinus Cinereus)的Cip1过氧化物酶基因(Cip1)，其大小在1.0~1.5kb之间。
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.