National Institute of Plant Genome Research, New Delhi, India.
Mol Biotechnol. 2013 Jun;54(2):133-40. doi: 10.1007/s12033-012-9549-0.
The Gateway(®) recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.
Gateway（®）重组技术彻底改变了基因克隆的方法，可用于功能分析和高通量 ORFeome 项目。通常情况下，Gateway 克隆的效率非常高，因为在 LR 重组和细菌转化后，只有含有重组目的克隆的细胞才能在抗生素选择平板上被选择出来。然而，当用于细菌选择的抗生素抗性基因在入口载体和目的载体中相同时，直接在抗生素平板上选择重组目的克隆是困难的。在这里，我们展示了一种在这种情况下直接在抗生素选择平板上获得阳性目的克隆的有效而全面的方法。该策略涉及使用结合在 attL 序列外的入口载体特异性引物对入口克隆进行聚合酶链反应（PCR）介导的扩增，然后使用该纯化的 PCR 产物与目的载体进行 LR 重组。我们的结果表明，通过 LR 重组将线性 DNA 片段克隆到圆形目的载体中是一种高效的方法，适用于大小达 7kb 的插入片段。使用这种方法，对于我们实验中测试的各种大小的基因，菌落 PCR 阳性目的克隆的产量为 100%。
