Autor(es): Zhang Pingping, Ding Yingying, Liao Wenting, Chen Qiuli, Zhang Huaqun, Qi Peipei, He Ting, Wang Jinhong, Deng Songhua, Pan Tianyue, Ren Hao, Pan Wei

Resumo: 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 - sequencing of four positive T-clones of the synthesised genes - (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming - secondary structure were found to be the principal obstacles preventing successful gene synthesis - were easily identified - solved in this method. Compensating for the disadvantages of being laborious - time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases - good allowance for Taq polymerase, oligonucleotides, PCR conditions - a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.

Palavras-Chave: Gene synthesis; Sequential OE-PCR; Oligonucleotides; Error correction

Imprenta: Gene, v. 524, n. 2, p. 347-354, 2013

Identificador do Objeto Digital: 10.1016/j.gene.2013.03.126

Descritores: Chikungunya virus - DNA ; Chikungunya virus - Genome ; Chikungunya virus - Molecular structure ; Chikungunya virus - Pathogenesis ; Chikungunya Virus - Virus

Data de Publicação: 2013