Key Laboratory of Non-coding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Mol Biotechnol. 2013 Sep;55(1):10-6. doi: 10.1007/s12033-012-9597-5.
PCR product cloning is the foundational technology for almost all fields in the life sciences. Numerous innovative methods have been designed during the past few decades. Enzyme-free cloning is the only one that avoids post-amplification enzymatic treatments, making the technique reliable and cost effective. However, the complementary staggered overhangs used in enzyme-free cloning tend to result in self-ligation of the vector under some circumstances. Here, we describe a "T-type" enzyme-free cloning method: instead of designing the complementary staggered overhangs used in conventional enzyme-free cloning, we create "T-type" overhangs that reduce the possibility of self-ligation and are more convenient for multi-vector cloning. In this study, we systematically optimize "T-type" enzyme-free cloning, compare its cloning background with that in conventional enzyme-free cloning, and demonstrate a promising application of this technique in multi-vector cloning. Our method simplifies post-amplification procedures and greatly reduces cost, offering a competitive option for PCR product cloning.
PCR 产物克隆是生命科学几乎所有领域的基础技术。在过去几十年中，设计了许多创新方法。无酶克隆是唯一一种避免扩增后酶处理的方法，使该技术可靠且具有成本效益。然而，无酶克隆中使用的互补交错突出端在某些情况下容易导致载体自身连接。在这里，我们描述了一种“T 型”无酶克隆方法：不是设计传统无酶克隆中使用的互补交错突出端，而是创建“T 型”突出端，从而降低自身连接的可能性，并且更便于多载体克隆。在这项研究中，我们系统地优化了“T 型”无酶克隆，比较了其与传统无酶克隆的克隆背景，并展示了该技术在多载体克隆中的有前途的应用。我们的方法简化了扩增后的步骤，大大降低了成本，为 PCR 产物克隆提供了一种有竞争力的选择。
