LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62
LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62
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DOI：
10.1007/s12161-012-9438-y
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发表时间：
2013-04-01
影响因子：
2.9
通讯作者：
Milcamps, Anne
中科院分区：
文献类型：
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作者：
Spalinskas, R.;Van den Bulcke, M.;Milcamps, Anne
有关转基因(S)在转基因生物中的插入位置和特征的信息对于转基因生物的安全性评估和鉴定非常重要。一般情况下，特别是在涉及转基因生物的紧急情况或快速警戒情况下，这种信息的产生大大得益于简单、高效和快速的方法的提供。在这里，我们报告了一种称为“基因组DNA末端快速扩增”(RADE)的非限制性内切酶方法的改进。该方法是以玉米事件MON810基因组DNA为模型系统，检测标准Taq聚合酶或聚合酶混合物(标准Taq聚合酶和校对TGO聚合酶(LT-RADE))。这两种方法都会产生最初的单链DNA，然后是嵌套的聚合酶链式反应步骤，并产生易于分离的DNA片段供进一步操作。我们发现，Taq/TGO聚合酶混合物的应用显著增加了所获得的PCR产物的大小。利用LT-RADE，我们成功地分离了转基因玉米事件MON810转基因插入片段的侧翼区域，并证实了该插入片段相邻区域的现有数据。此外，应用我们的方法可以首次有效地分离和鉴定转基因水稻事件LLRICE62插入周围的DNA序列。
Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information in general and in particular in emergencies or rapid alert situations involving GMO greatly benefit from the availability of simple, efficient, and rapid approaches. Here, we report on the improvement of a restriction independent method named "Rapid Amplification of genomic DNA Ends" (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE)). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE, we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, application of our approach allowed to efficiently isolate and identify, for the first time, the DNA sequences surrounding the insert of GM rice event LLRICE62.