An Improved Method of Gene Synthesis Based on DNA Works Software and Overlap Extension PCR

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.

Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

“…The designed chimeric riboswitch was amplified from primers ID363 to ID376 and inserted into the EcoRI and BglII sites of pAM001, a vector containing GFP and a spectinomycin resistance cassette flanked by sequences from B. subtilis amyE, using isothermal assembly (51,52,56). The pAM001 plasmid was generated for this work via insertion of annealed primers AM005 and AM006 into pMF35 (54) linearized at EcoRI and HindIII sites to introduce a multiple-cloning site with NheI, SpeI, and SphI restriction sites.…”

Hypoosmotic Expression of Dunaliella bardawil ζ-Carotene Desaturase Is Attributed to a Hypoosmolarity-Responsive Element Different from Other Key Carotenogenic Genes

“…nih.gov/dnaworks/). The ble coding sequence synthesis condition was as described by Dong et al (2007a). The chimeric fragment of ble-egfp with AscI and SacII restriction sites in the corresponding ends was obtained by means of a two-step overlap PCR method (Heckman and Pease, 2007).…”

Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

“…The third factor is the gene synthesis of ZFPs. The gene synthesis method used was a reproducible simple method of less error based on DNAWorks (Dong 2007). The genes could be synthesized using two-step PCR, including the overlap extension (OE) PCR and amplification PCR.…”

“…To construct the coding sequences of ZFPs, the two-step PCR method, overlap extension (OE) PCR and amplification PCR, was used for gene synthesis according to Dong et al (Dong, 2007). Fourteen oligonucleotides were mixed and diluted to 2 µM with ddH 2 O, which were used as the primer mixture in the PCR for synthesizing gene.…”