Figure 2

Scheme for the generation of cohesive ends. In addition to regions complementary to the insert DNA sequence (1), oligonucleotides are designed with overhangs comprising the 4 bp cohesive part of a restriction site combined with deoxyinosine (dI) at the third position from the 5′ end. Primer annealing and extension during a PCR leads to amplification of the desired target fragment (2), which carries the dI residues (bold, orange) in its termini. The pairing properties of the universal base will generate a sequence distribution at the corresponding site of the opposing strand (indicated as 'N’). Purified PCR products are treated with EndoV, which cleaves the second phosphodiester bond 3′ to dI (3). The target DNA fragment (4) is obtained by spin column-based or agarose gel purification, respectively, removing the weakly bound residues of ssDNA. Carrying cohesive ends with 5′ phosphates, the insert fragment is now suitable for ligation to vector DNA fragments created by conventional restriction enzyme treatment (in the depicted case SacI and KpnI).