High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides.
High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides.
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DOI：
10.1093/nar/gkq677
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发表时间：
2010-10
影响因子：
14.9
通讯作者：
Sykes KF
中科院分区：
文献类型：
--
作者：
Borovkov AY;Loskutov AV;Robida MD;Day KM;Cano JA;Le Olson T;Patel H;Brown K;Hunter PD;Sykes KF
为了满足日益增长的合成基因的需求，必须开发更强大，可扩展和廉价的基因组装技术。在这里，我们提出了一个高质量的基因组装协议，直接从低成本的边缘质量的微阵列合成的寡核苷酸。值得注意的是，我们通过使用嵌入在组装过程中的基于杂交的选择消除了耗时和费钱的寡核苷酸纯化步骤。该协议进行了测试，高达2000寡核苷酸的混合物直接从微阵列从三个不同的芯片制造商获得洗脱。这些混合物含有<5%的完美寡核苷酸，并直接用于组装27个不同大小的测试基因。通过测序评估基因质量，并在偶联的体外转录/翻译反应中测试其活性。使用新方案从微阵列洗脱材料组装的基因与通过标准方案从>95%纯的柱合成寡核苷酸组装的基因的质量相匹配。这两个平均只有2.7错误/kb，基因组装从微阵列洗脱材料没有克隆选择产生的蛋白质只有30%，比序列确认的克隆。这份报告代表了第一次演示的成本效益的基因组装从微阵列合成的寡核苷酸。通过这种方法组装的总成本接近每个碱基5美分，使基因合成比传统的克隆更便宜。
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.
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影响因子：
14.9
作者：
Egeland RD;Southern EM
通讯作者：
Southern EM
DOI：
10.1073/pnas.0803240105
发表时间：
2008-07-08
影响因子：
11.1
作者：
Krishnakumar, Sujatha;Zheng, Jianbiao;Davis, Ronald
通讯作者：
Davis, Ronald
DOI：
10.1006/bbrc.1998.8929
发表时间：
1998-07-09
影响因子：
3.1
作者：
Au, LC;Yang, FY;Kao, CF
通讯作者：
Kao, CF
影响因子：
3.5
作者：
STEMMER, WPC;CRAMERI, A;HEYNEKER, HL
通讯作者：
HEYNEKER, HL
影响因子：
10.7
作者：
Henry, Ian;Sharp, Paul M.
通讯作者：
Sharp, Paul M.