Autosticky PCR: Directional Cloning of PCR Products with Preformed 5 Overhangs

The polymerase chain reaction (PCR) is a method of central importance in molecular biology (1 ,2 ). The DNA fragment of interest is often amplified for cloning purposes. A frequently used experimental approach is to include extra restriction endonuclease cleavage sites in the amplification primers, digestion of the PCR product with the corresponding enzymes, and ligation of the product to a linearized cloning vector (3 ). However, the efficiency of cleavage by certain restriction endonucleases is rather low because of the cleavage site(s) being too close to the termini of a DNA fragment (4 ,5 ), and internal restriction sites of the fragment might also complicate the cloning.