Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA
Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA
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DOI：
10.2144/99272st01
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发表时间：
1999-08-01
期刊：
影响因子：
2.7
通讯作者：
Bellemare, G
中科院分区：
文献类型：
--
作者：
Spertini, D;Béliveau, C;Bellemare, G
为了筛选转基因植物材料中的转移DNA（T-DNA）整合，我们发展了一种基于特异性扩增T-DNA边界侧翼的植物基因组DNA的方法。这种方法是可能的，因为限制性片段上T-DNA末端侧翼区域的长度对整合基因座是特异性的。我们已经修改了接头连接PCR技术开发的未知DNA侧翼已知序列的扩增。获得的PCR模式是特定的和可重复的不同植物从一个给定的转基因系。此外，获得的PCR产物的数量可以被认为是T-DNA拷贝数的良好估计。当与Southern印迹分析相比时，PCR结果提供了关于T-DNA整合模式的复杂性以及关于T-DNA边界的完整性的有价值的补充信息。我们描述了这种方法的应用程序的转基因拟南芥植物的群体。
For the screening of transfer DNA (T-DNA) integration in transgenic plant material, we developed a method based on specific amplification of genomic plant DNA flanking T-DNA borders. This approach is possible because the length of the region flanking T-DNA extremity on a restriction fragment is specific to the integration locus. We have modified an adaptor ligation PCR technique developed for amplification of unknown DNA flanking known sequence. The PCR patterns obtained were specific and reproducible for different plants from a given transgenic line. Furthermore, the number of PCR products obtained could be considered a good estimation of the T-DNA copy number. When compared to Southern blot analysis, the PCR results give valuable complementary information about the complexity of the T-DNA integration pattern and also about the integrity of the T-DNA borders. We describe the applications of this approach to populations of transgenic Arabidopsis thaliana plants.