全基因合成方法学研究

通过基因全合成实现基因的分子改造和人工组建正成为一种实验室常规手段,因此,建立一种能够在相对低廉和短时间内准确和高效地设计和合成长片段基因的方法十分重要.本研究报道了一种重复性好、错误率低、低成本和简便的基因设计和全合成方法.此方法包含经DNA2.0软件的序列优化,Gene2Oligo软件的寡核苷酸设计,覆盖全长基因双链的寡核苷酸的组合和引物介导下的全基因的合成等步骤.运用此方法,对3个不同长度的基因(分别为653bp,1309bp和1498bp)成功地进行了密码子优化和一步全合成.其中的amyFF在大肠杆菌中表达量提高了12倍.图2参18

Methodological Studies on Total Gene Synthesis

Gene modification or artificial construction through in vitro total gene synthesis is becoming more acceptable, and the ability to efficiently and accurately synthesize long DNA sequences at low cost and in short time is, therefore, becoming increasingly important. In this work, a simple, reproducible, less error-prone and cost-effective protocol for total gene design and synthesis was described. It was a combination of DNA sequence optimization by DNA2.0, oligonucleotides design by Gene2Oligo, the complete sequence of both strands-covered oligonucleotides assembly and total gene synthesis by primer-mediated amplification. Based on this protocol, three genes with different lengths (653 bp, 1 309 bp and 1 498 bp) were successfully codon-modified and one-step synthesized. The expression efficiency of codon-optimized amyFF was increased by 12 times in Escherichia coli. Fig 2, Ref 18