Improvement And Verification The Gene Synthesis Method

In the post-genomic era,with development of research about protein structure and function, rare codon and high level expression, nucleic acid vaccine, gene therapy, the ability to synthesize any arbitrary DNA sequence is increasingly in demand. The major drawback of total gene synthesis is the high tendency of DNA sequence errors and the high cost with all current gene synthesis methods.In this study, we describe an improved total gene synthesis strategy from two aspects of cost and base error rate. The optimal length of oligonucleotide synthesized by chemical method is 55-59bp, the overlapping region is 15–18 bases, the Tm of overlaps could not lower than 50℃. DA-PCR is carried out for every four not more than six consecutive oligoucleotides, each inner oligonucleotide concentration in the reaction mixture is 25nM, and outer oligonucleotide is 200 nM, using touchdown PCR in annealing steps. Production of DA-PCR and OE-PCR were treated with T7 endonuclease I is presented in the synthesis procedures to separate the DNA contained base errors. Using all of these measures in the synthesis strategy can reduce the error rate greatly, the synthesis cost and workload can reduces significantly at the same time, all this is proved by Escherichia coli codon optimized hGH gene,human IL-18 gene and Pichia pastoris codon optimized G-CSF gene synthesised.The Escherichia coli codon bias optimized hGH gene was 1040 bp in length and dissected into 30 oligonucleotides of 58 nt each. The product of two synthesis step were treated with T7 endonuclease I respectively. The sequencing results shown that in the control group which was not treated with T7 endonuclease I, none of the four clones was the accurate sequence. There was total 33 base errors on whole 4158 nucleotides. In four of the clones which was treated with T7endonuclease I,two of them were confirmed to be the correct products. At the same time,the error rate of the whole sequence of the four positive clones was reduced to a very low level,only 2 base errors on whole 4159 nucleotides.The codon preference of E.coli optimized human IL-18 gene is 495 bp,designed and synthesized 18 oligonucleotides. The whole DNA sequence was synthesized by an one-step and two-step total gene synthesis method respectively, and then inserted to pUC18 vector, two positive clones of each method were sended to sequence, the result showed that one clones was correct completely in all four positive clones.Pichia pastoris bias optimized G-CSF gene is 546 bp in length and dissected into 16 oligonucleotides of 56 nt each and the overlapping region was 18–20 bases, synthesized by one step. The sequencing results shown that the six clones didn′t treat with T7 endonuclease I have none of the accurate sequence. There was total 17 base errors on whole 3212 nucleotides. Four of the six clones treated with T7endonuclease I were confirmed to be the correct products. At the same time,the error rate was reduced to a very low level,only 2 base error on whole 3211 nucleotides.Although mass clone sequencing can found the clone contains completely correct gene, but in some case it cann′t achieve their aims. In this paper we put forward a methods to repair the base errors in the sequencing clone. Oligonucleotide coverage mothod(OCM) can repair the errors in any site directly, is a smart gesign to solve problems of base errors.