Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process.
Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process.
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DOI：
10.1093/nar/gkq163
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发表时间：
2010-05
影响因子：
14.9
通讯作者：
Caruthers MH
中科院分区：
文献类型：
--
作者：
LeProust EM;Peck BJ;Spirin K;McCuen HB;Moore B;Namsaraev E;Caruthers MH
通过在微阵列上平行合成 DNA，我们已经能够以 fmol 的量合成数千种独特的长寡核苷酸 (150mers)。在如此大规模的合成中，寡核苷酸的序列准确性受到所用 DNA 合成过程的产量和副反应的限制。虽然对长寡核苷酸文库（150mer 及以上）的需求很大，但传统 DNA 合成的产量和相关的副反应此前已将寡核苷酸库的可用性限制为长度 <100 nt。使用基于新型阵列的脱嘌呤测定，我们发现脱嘌呤副反应是在安捷伦科技公司的 SurePrint® DNA 微阵列平台上合成长寡核苷酸文库的限制因素。我们还展示了如何通过新型脱三苯甲基化工艺来控制和减少脱嘌呤，从而合成高质量的长（150mer）寡核苷酸文库，并报告了此类文库的合成效率的表征。用这种方法制备的寡核苷酸文库改变了几种现有应用（例如靶向重测序、shRNA文库的制备、定点诱变）的经济性和可用性，并且有可能实现更多新颖的应用（例如高复杂性合成生物学）。
We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies’ SurePrint® DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).
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影响因子：
46.9
作者：
Ball, Madeleine P.;Li, Jin Billy;Gao, Yuan;Lee, Je-Hyuk;LeProust, Emily M.;Park, In-Hyun;Xie, Bin;Daley, George Q.;Church, George M.
通讯作者：
Church, George M.
影响因子：
46.9
作者：
Deng, Jie;Shoemaker, Robert;Xie, Bin;Gore, Athurva;LeProust, Emily M.;Antosiewicz-Bourget, Jessica;Egli, Dieter;Maherali, Nimet;Park, In-Hyun;Yu, Junying;Daley, George Q.;Eggan, Kevin;Hochedlinger, Konrad;Thomson, James;Wang, Wei;Gao, Yuan;Zhang, Kun
通讯作者：
Zhang, Kun
影响因子：
56.9
作者：
CARUTHERS, MH
通讯作者：
CARUTHERS, MH
DOI：
10.1073/pnas.0702165104
发表时间：
2007-05-29
影响因子：
11.1
作者：
Dahl, Fredrik;Stenberg, Johan;Ji, Hanlee
通讯作者：
Ji, Hanlee
影响因子：
48
作者：
Cleary, MA;Kilian, K;Hannon, GJ
通讯作者：
Hannon, GJ