One-Pot Preparation of mRNA/cDNA Display by a Novel and Versatile Puromycin-Linker DNA
One-Pot Preparation of mRNA/cDNA Display by a Novel and Versatile Puromycin-Linker DNA
复制标题
DOI：
10.1021/co2000295
复制
发表时间：
2011-09-01
影响因子：
--
通讯作者：
Nemoto, Naoto
中科院分区：
文献类型：
--
作者：
Mochizuki, Yuki;Biyani, Manish;Nemoto, Naoto
提出了一种新设计的嘌呤霉素连接DNA的mRNA/cDNA展示的快速、简便和稳健的制备方法。这种新的接头结构简单，易于合成，并且成本效益高，可用于“体外肽和蛋白质选择”。将RNase T1核酸酶位点引入到新的接头中有利于mRNA/cDNA的容易回收。通过提高接头与mRNA的连接效率和mRNA/cDNA展示蛋白质从固相（磁珠）的有效释放来展示蛋白质。对于应用程序演示，成功执行了亲和性选择。此外，我们还引入了一种“一锅法”制备方案，以方便地进行mRNA展示。与传统的方法，需要繁琐的和下游的多步骤的过程，包括纯化，该协议将使mRNA/cDNA展示方法更实用和方便，也有利于下一代，高通量mRNA/cDNA展示系统的发展服从自动化。
A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA. displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.