In recent years, many novel approaches to molecular cloning have been proposed to expedite the procedure, enhance cloning efficiency and bypass the requirement of restriction sites. Despite its widespread use, the traditional, restriction-ligation-based cloning protocol suffers from major problems, including, but not limited to: (i) low efficiency, (ii) dependency on the availability of unique restriction sites in a cloning vector and in the gene of interest, and (iii) time-consuming and labour-intensive process. Gene cloning is an indispensable molecular biology technique that, since its first introduction, has been central to the development of genetic engineering and, consequently, the entire field of life sciences. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists. QuickStep-Cloning expands the versatility of megaprimer-based cloning. coli cells prepared using the conventional calcium chloride method. The method is compatible with competent E. 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. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3′-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. 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. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Molecular cloning is an essential step in biological engineering.
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