DIRECTIONAL CLONING OF BLUNT-ENDED PCR PRODUCTS

A method that allows the directional cloning of blunt-ended polymerase chain reaction (PCR) fragments is described. One PCR primer must be 5 ' phosphorylated Extra bases are not required on either PCR primer. A linearized vector is enzymatically processed to contain a single 5'-te rminal phosphate. The monophosphorylated vector is amenable to recombi nant-insertion during ligation when the fragment is in the correct ori entation. Increased recombinant yield results from incubating the mono phosphorylated vector with a restriction enzyme (SrfI) that relineariz es nonrecombinant plasmids during the ligation reaction.