Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
IUBMB Life. 2012 Oct;64(10):841-5. doi: 10.1002/iub.1069.
In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3' end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications.
在本文中，我们开发了一种新的 PCR 方法，称为环连接 PCR，用于分离转基因作物中的侧翼序列。该方法的新颖之处在于它使用茎环结构来设计环连接接头。该接头设计为在与受限 DNA 连接时形成一个缺口。这种修饰不仅可以防止接头的自身连接，而且还可以促进环连接接头的 3'端的延伸，从而在连接产物中产生茎环结构。此外，茎环结构的抑制作用降低了非特异性扩增并提高了该方法的成功率；除了包含特定引物结合位点的连接产物之外，所有延伸产物将抑制指数扩增。使用该方法，分别从转基因玉米 LY038、DAS-59122-7、Event 3272 和转基因大豆 MON89788 中获得了 442、1830、107 和 512bp 的左边界侧翼序列。实验结果表明，环连接 PCR 是一种高效、可靠且具有成本效益的方法，可用于鉴定转基因作物中的侧翼序列，并且可应用于其他基因组步移应用。
