TOPO tools (PCMV/TetO 5′ element) for producing linear DNA constructs with defined 5′ and 3′ functional elements. (A) The 5′ and 3′ functional elements and the PCR product to which they are to be added are shown. Different 11-bp sequences (bold) are added to the ends of the PCR product via forward and reverse primers. (B) TOPO cleaves the PCR product, creating TOPO-activated overhangs (and the release of single-stranded 6-mers) complementary to the overhangs of the TOPO-activated functional elements. (C) Annealing the overhangs juxtaposes the 5′-OH of each to attack the phosphotyrosyl bond of the other, ligating both strands of each functional element to both strands of the PCR product, releasing two TOPO molecules, and creating a linear recombinant template for direct use in vitro and in vivo. (Reproduced from Invitrogen 2002, part of Life Technologies, 2002.)
