Removing Restrictions from Molecular Cloning

Traditional molecular cloning techniques focus on using a variety of restriction enzymes combined with DNA ligase to generate plasmids containing recombinant DNA. By contrast, more modern techniques utilize overlapping, homologous ends eliminating the need for restriction enzymes and reducing the number of steps required to generate a functional construct. Aided by the development of standardized parts, we now have the ability to construct plasmids and vectors faster, more reliably and with a greater variety than ever before. This technology enables cloning and optimization on a combinatorial scale, mixing and matching parts such as promoters, tags and selection markers, and provides extensive options for a range of applications. We will compare several next-gen DNA assembly technologies, demonstrate the sizable advantage with combinatorial cloning when trying to express difficult proteins, and discuss the importance of standardized components to efficient DNA assembly,