A simple,universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis |
| |
Authors: | Pingping Zhang Yingying Ding Wenting Liao Qiuli Chen Huaqun Zhang Peipei Qi Ting He Jinhong Wang Songhua Deng Tianyue Pan Hao Ren Wei Pan |
| |
Affiliation: | 1. Department of Medical Microbiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China;2. Department of Physiology, Anhui Medical University, Hefei 230032, China;3. Shanghai Medical College, Fudan University, Shanghai 200032, China |
| |
Abstract: | 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. |
| |
Keywords: | OE-PCR, overlap extension polymerase chain reaction Tm, melting temperature oligos, oligonucleotides nt, nucleotide |
本文献已被 ScienceDirect 等数据库收录! |
|