Figure 2.

Ligation-independent cloning using pHG7: (1) An ORF target is amplified using primers with extensions homologous to the DNA ends generated by linearization of the expression vector. After amplification (2), the PCR product is treated with T4 DNA polymerase in the presence of one dNTP (dATP in this example). (3) 3′-5′ exonuclease activity results in a 5′ overhang and a terminal dATP on the opposite strand. This product can be annealed (4) to similarly treated plasmid DNA (with dTTP the only included dNTP) to generate a recombinant plasmid upon transformation (5).