Hommelsheim Carl Maximilian, Frantzeskakis Lamprinos, Huang Mengmeng, Ülker Bekir
Plant Molecular Engineering Group, IZMB (Institute of Cellular and Molecular Botany), University of Bonn, Kirschallee 1, 53115 Bonn.
1] Plant Molecular Engineering Group, IZMB (Institute of Cellular and Molecular Botany), University of Bonn, Kirschallee 1, 53115 Bonn [2].
Sci Rep. 2014 May 23;4:5052. doi: 10.1038/srep05052.
Designer transcription-activator like effectors (TALEs) is a promising technology and made it possible to edit genomes with higher specificity. Such specific engineering and gene regulation technologies are also being developed using RNA-binding proteins like PUFs and PPRs. The main feature of TALEs, PUFs and PPRs is their repetitive DNA/RNA-binding domains which have single nucleotide binding specificity. Available kits today allow researchers to assemble these repetitive domains in any combination they desire when generating TALEs for gene targeting and editing. However, PCR amplifications of such repetitive DNAs are highly problematic as these mostly fail, generating undesired artifact products or deletions. Here we describe the molecular mechanisms leading to these artifacts. We tested our models also in plasmid templates containing one copy versus two copies of GFP-coding sequence arranged as either direct or inverted repeats. Some limited solutions in amplifying repetitive DNA regions are described.
设计转录激活因子样效应物（TALEs）是一项很有前景的技术，它使得以更高的特异性编辑基因组成为可能。利用像PUFs和PPRs这样的RNA结合蛋白，也正在开发此类特定的工程和基因调控技术。TALEs、PUFs和PPRs的主要特征是它们具有单核苷酸结合特异性的重复DNA/RNA结合结构域。如今可用的试剂盒使研究人员在生成用于基因靶向和编辑的TALEs时，能够以他们想要的任何组合组装这些重复结构域。然而，此类重复DNA的PCR扩增存在很大问题，因为这些扩增大多会失败，产生不想要的假象产物或缺失。在这里，我们描述了导致这些假象的分子机制。我们还在含有一份或两份以正向或反向重复排列的绿色荧光蛋白编码序列的质粒模板中测试了我们的模型。文中描述了一些扩增重复DNA区域的有限解决方案。
