title: A simple, universal, efficient PCR-based gene synthesis method: sequential OE-PCR gene synthesis.
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.
authors.affiliation: Department of Medical Microbiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Zhang
authors.forename: Yingying
authors.initials: Y
authors.lastname: Ding
authors.forename: Wenting
authors.initials: W
authors.lastname: Liao
authors.forename: Qiuli
authors.initials: Q
authors.lastname: Chen
authors.forename: Huaqun
authors.initials: H
authors.lastname: Zhang
authors.forename: Peipei
authors.initials: P
authors.lastname: Qi
authors.forename: Ting
authors.initials: T
authors.lastname: He
authors.forename: Jinhong
authors.initials: J
authors.lastname: Wang
authors.forename: Songhua
authors.initials: S
authors.lastname: Deng
authors.forename: Tianyue
authors.initials: T
authors.lastname: Pan
authors.forename: Hao
authors.initials: H
authors.lastname: Ren
authors.forename: Wei
authors.initials: W
authors.lastname: Pan
mesh_terms: D000595:Amino Acid Sequence; D002646:Chikungunya virus / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000138:chemical synthesis* / Q000737:chemistry; D029821:Enterovirus A, Human / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005813:Genes, Synthetic; D005814:Genes, Viral; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013997:Time Factors
chemical_list: D004279:DNA, Viral; D009841:Oligonucleotides