Joint International Research Laboratory of Metabolic and Developmental Sciences, National Center for Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Mol Biotechnol. 2019 Sep;61(9):694-702. doi: 10.1007/s12033-019-00196-0.
Molecular characteristics including information of insertion site, flanking sequence, and copy numbers are the base for the safety assessment and subsequent monitoring of genetically modified organisms (GMOs), which has to be revealed thoroughly in a case-by-case manner. Although both polymerase chain reaction (PCR)-based and next-generation sequencing (NGS)-based approaches are proven to be effective in the molecular characterization of most of GM events, they often fail to work with GM maize events, mainly due to the genome complexity. In this study, by using NGS, we successfully identified the 3' end T-DNA insertion site and flanking sequence of a GM maize event IE09S034, which were confirmed by PCR amplification and Sanger sequencing. Notably, insertions of unintended exogenous elements were revealed in this event although the single copy of target exogenous genes was also confirmed by digital PCR. The output of this study provides novel and important genetic evidence for the safety assessment and monitoring of GM maize event IE09S034.
分子特征，包括插入位点、侧翼序列和拷贝数，是转基因生物（GMO）安全性评估和后续监测的基础，必须逐个案例进行彻底揭示。尽管基于聚合酶链反应（PCR）和基于下一代测序（NGS）的方法已被证明在大多数 GM 事件的分子特征描述中是有效的，但它们通常无法用于 GM 玉米事件，主要是由于基因组的复杂性。在本研究中，我们通过使用 NGS，成功地鉴定了 GM 玉米事件 IE09S034 的 3'末端 T-DNA 插入位点和侧翼序列，这些结果通过 PCR 扩增和 Sanger 测序得到了验证。值得注意的是，尽管通过数字 PCR 也证实了靶标外源基因的单拷贝，但在该事件中也揭示了非预期的外源元件的插入。本研究的结果为 GM 玉米事件 IE09S034 的安全性评估和监测提供了新的、重要的遗传证据。
