A simple, universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis

作者：Zhang Pingping; Ding Yingying; Liao Wenting; Chen Qiuli; Zhang Huaqun; Qi Peipei; He Ting; Wang Jinhong; Deng Songhua; Pan Tianyue; Ren Hao^*; Pan Wei

来源：Gene, 2013, 524(2): 347-354.

DOI：10.1016/j.gene.2013.03.126

• Gene synthesis
• Sequential OE-PCR
• Oligonucleotides
• Error correction

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.

• 出版日期2013-7-25
• 单位安徽医科大学; 中国人民解放军第二军医大学; 复旦大学