title: QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: ChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK.
authors.forename: Pawel
authors.initials: P
authors.lastname: Jajesniak
authors.affiliation: ChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD UK.
authors.forename: Tuck Seng
authors.initials: TS
authors.lastname: Wong
keywords: Directed evolution; Gene cloning; Megaprimer; Metabolic engineering; Molecular cloning; Protein engineering; Recombinant DNA; Recombinant plasmid; Synthetic biology