International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Anal Biochem. 2011 Sep 15;416(2):196-201. doi: 10.1016/j.ab.2011.05.021. Epub 2011 May 24.
Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.
从众多疑似转基因生物中鉴定出一个良好的转基因事件对于进一步的特征描述至关重要。在转基因植物中，转基因可以通过破坏内源性基因和/或在异染色质区域整合到单个或多个位置，从而导致位置效应。除此之外，为了防止转基因植物的非法使用，每个商业转基因事件的转基因整合特征都需要进行鉴定。在这里，我们展示了一种基于亲和性的基因组步行方法，命名为定位发现（LF）PCR（聚合酶链式反应），用于确定农杆菌转化的水稻植株的转基因侧翼序列。LF-PCR 包括一个由简并引物和转移 DNA（T-DNA）特异性引物进行的初级 PCR，一个嵌套 PCR，以及一种通过使用与 T-DNA 互补的生物素标记引物来富集所需扩增子的方法。这种富集技术将所需扩增子的单链与非靶扩增子分离，模板复杂度降低了几个数量级。我们分析了 8 个转基因水稻植株，在 3 个不同的染色体上找到了转基因的整合位点。还从测序的整合位点观察到了农杆菌属的特征性非同源重组。我们相信 LF-PCR 应该成为转基因分析中不可或缺的技术。
