Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis
Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis
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DOI：
10.1038/nmeth724
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发表时间：
2004-12-01
期刊：
影响因子：
48
通讯作者：
Hannon, GJ
中科院分区：
文献类型：
--
作者：
Cleary, MA;Kilian, K;Hannon, GJ
定义的核酸序列的复杂文库生成可以极大地帮助蛋白质和基因功能的功能分析。以前，此类研究依赖于单独合成的Otigonuctepides或细胞核酸作为起始材料。由于每种方法都有缺点，因此我们开发了一种快速且成本效益的替代方案，用于构建定义序列的小型碎片DNA文库。这种方法使用原位微阵列DNA合成来产生复杂的寡核苷酸群体。这些人群可以被恢复并直接使用或通过克隆永生。从单个微阵列中，可以生成一个包含数千个独特序列的库。作为这项技术潜在应用的一个例子，我们已经测试了针对许多人类和小鼠基因的短发夹RNA（SHRNA）生产质粒的方法。我们以统一的图书馆序列表示高保真克隆的检索。
Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized otigonucteotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.