PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets.
PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets.
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DOI：
10.1093/nar/gni143
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发表时间：
2005-10-06
影响因子：
14.9
通讯作者：
Nakano H
中科院分区：
文献类型：
--
作者：
Kojima T;Takei Y;Ohtsuka M;Kawarasaki Y;Yamane T;Nakano H
我们开发了一种基于固相单分子PCR在油包水（W/O）乳液中形成的精细且稳健的水相区室中的磁性微珠上构建基因文库的新方法。在该方法中，将严格稀释的DNA片段作为模板分布在乳液上，其中与多个引物和其他PCR组分交联的珠粒被封装以形成多个反应隔室。然后将递送的DNA扩增并共价固定在单独隔室内的平行珠上，以构建珠上遗传文库（地球仪），其容易适用于由定义的DNA结合蛋白识别的遗传元件的全基因组全局扫描。我们构建了一个地球仪的Paracoccus ficans和选择的基因珠结合到His标记的转录因子PhaR的流式细胞术。作为用抗His荧光抗体进行流式细胞术筛选的结果，用该系统从该文库富集了1200倍的PhaR靶片段。因此，该系统是在全基因组范围内分析转录网络的有力工具。
We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.
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影响因子：
13.8
作者：
Orlando, V
通讯作者：
Orlando, V
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作者：
Doi, N;Yanagawa, H
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