Center for Innovations in Medicine of the Biodesign Institute at the Arizona State University, Tempe, AZ 85287, USA.
Nucleic Acids Res. 2010 Oct;38(19):e180. doi: 10.1093/nar/gkq677. Epub 2010 Aug 6.
To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning.
为了满足对合成基因日益增长的需求，必须开发更强大、可扩展和更经济的基因组装技术。在这里，我们提出了一种从低成本、低质量的微阵列合成寡核苷酸直接进行高质量基因组装的方案。值得注意的是，我们通过在组装过程中嵌入基于杂交的选择，消除了耗时且昂贵的寡核苷酸纯化步骤。该方案在直接从三个不同芯片制造商获得的微阵列洗脱的多达 2000 个寡核苷酸的混合物上进行了测试。这些混合物含有<5%的完美寡核苷酸，并直接用于 27 个不同大小的测试基因的组装。通过测序评估基因质量，并在体外转录/翻译偶联反应中测试其活性。使用新方案从微阵列洗脱材料组装的基因与使用标准方案从>95%纯柱合成寡核苷酸组装的基因质量相当。两者的平均错误率仅为 2.7/kb，并且未经克隆选择从微阵列洗脱材料组装的基因产生的蛋白质比经序列确认的克隆少 30%。本报告代表了首次从微阵列合成寡核苷酸进行经济高效的基因组装的演示。该方法的组装总成本接近每个碱基 5 美分，使基因合成比传统克隆更具成本效益。
