Directional cloning of PCR products.

INTRODUCTION This protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction (Liu and Schwartz 1992) to relinearize any self-religating vector DNA. Directional cloning is achieved by creating a monophosphorylated vector and a monophosphorylated insert. In the desired orientation, ligation results in a single-nicked, circular molecule. In the undesired, opposite orientation, the ligation results in a linear molecule that transforms E. coli with a drastically reduced efficiency. A monophosphorylated vector is created by enzymatically treating the vector with a restriction endonuclease, removing the exposed 5' phosphates with an alkaline phosphatase, and subsequently digesting the vector with a second restriction endonuclease. A monophosphorylated insert is created using one 5'-phosphorylated primer that can be either machine-synthesized or obtained by kinase treatment.