DNA Cloning

DNA cloning is a core technique in biomedical and biotechnological research and is used to assemble and modify DNA fragments at will. While DNA cloning has traditionally relied on restriction enzymes, recent homology-based methods offer improved protocols together with seamless and directional assembly of desired products, overcoming the main disadvantages of restriction enzyme DNA cloning. This chapter provides a historical perspective on DNA cloning, presents a detailed discussion on state-of-the-art in vitro and in vivo homology-based methodologies, covering the basics of how to perform all major plasmid modifications (sub-cloning, site-directed mutagenesis, insertions, and deletions), and gives examples of how to apply these techniques for complex DNA cloning projects.