Agilent Technologies Inc., LSSU - Genomics, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, USA.
Nucleic Acids Res. 2010 May;38(8):2522-40. doi: 10.1093/nar/gkq163. Epub 2010 Mar 22.
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).
我们已经实现了在微阵列上进行平行 DNA 合成，以毫微微摩尔量合成数千种独特的长寡核苷酸（150 个碱基）的能力。在如此大规模的合成中，寡核苷酸的序列准确性受到所使用的 DNA 合成过程的产率和副反应的限制。虽然对长寡核苷酸（150 个碱基及以上）文库的需求很大，但传统 DNA 合成的产率和相关的副反应以前限制了寡核苷酸库的可用性，使其长度<100 个碱基。我们使用新型基于阵列的脱嘌呤测定法，表明脱嘌呤副反应是在安捷伦科技的 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的脱保护过程来控制和减少脱嘌呤反应，从而能够合成高质量的长（150 个碱基）寡核苷酸文库，并报告了这种文库的合成效率的特征。使用这种方法制备的寡核苷酸文库改变了几个现有应用的经济性和可用性（例如靶向重测序、shRNA 文库的制备、定点诱变），并且有可能实现更多新的应用（例如高复杂度的合成生物学）。
