QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids

Background Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. Result Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3′-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. Conclusion QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists. Electronic supplementary material The online version of this article (doi:10.1186/s13036-015-0010-3) contains supplementary material, which is available to authorized users.