Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

A novel method for the directional cloning of native PCR products was devel oped. A basic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5' ends of PCR pro duct strands contain built-in amplification primers, abasic sites within th e primers result in the formation of 5' single-stranded overhangs at the en ds of the PCR product, enabling its direct ligation to a suitably cleaved c loning vector without any further modification. This "autosticky PCR" (AS-P CR) overcomes the problems caused by end sensitivity of restriction enzymes , or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5' overhang.