Gateway recombination-based cloning, which eliminates the use of restriction endonucleases and ligase, has been widely used for the construction of high-throughput (HTP) vectors. However, this approach is very expensive and its two-stage reaction process is laborious and time consuming. Therefore, we developed a Gateway cloning method that uses fusion-PCR to generate attL recombination site adaptors, and the PCR products, which can be directly cloned into destination vectors, giving rise to Rapid One-Step Gateway (ROG) Cloning. 100% of cloning efficiencies were obtained by this ROG method. This method has no BP reaction/entry clone step, thus halving the cost and time consumed. Overall, this work provides a highly efficient, rapid, low-cost method for directional recombination cloning.

中文翻译：

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Fusion-PCR生成attL重组位点适配器，并允许快速一步网关（ROG）克隆。

基于网关重组的克隆消除了限制性核酸内切酶和连接酶的使用，已被广泛用于构建高通量（HTP）载体。但是，该方法非常昂贵，并且其两阶段反应过程费力且费时。因此，我们开发了一种使用融合PCR生成atL重组位点衔接子的Gateway克隆方法，以及可以直接克隆到目的载体的PCR产物，从而产生了快速一步网关（ROG）克隆。通过这种ROG方法获得了100％的克隆效率。该方法没有BP反应/进入克隆步骤，因此使成本和时间减少了一半。总的来说，这项工作为定向重组克隆提供了一种高效，快速，低成本的方法。