Abstract
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.
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Acknowledgments
The authors thank Dr. Zhongdao Li, Shiqiang Lin, Liwei Wang, and Jinjun Wu for their comments and suggestions; Dr. J.E. Fleming for careful proofreading and revision of the manuscript. The authors thank Dr. Mingzhang Yang and Shanghua Fan for providing some of the experimental materials. This study was financially supported by the National Natural Science Foundation of China (Grant No: 31170132), National Basic Research Program of China (Grant No.: 2012CB518700).
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Yang, RB., Bi, LJ. & Zhang, XE. A Novel T-type Overhangs Improve the Enzyme-Free Cloning of PCR Products. Mol Biotechnol 55, 10–16 (2013). https://doi.org/10.1007/s12033-012-9597-5
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DOI: https://doi.org/10.1007/s12033-012-9597-5