Figure 1

Cohesive dsDNA ends created in this study. In order to ligate insert DNA fragments efficiently with a linearized target plasmid vector, both molecules have to carry compatible cohesive ends. For the vector DNA, 5′ recessed ends are created by conventional restriction enzyme treatment. Names and recognition sequences of the enzymes used in this study are listed. For other enzymes, please refer to REBASE [60]. Endonucleolytic cleavage positions are depicted as vertical dashes. Insert DNA fragments with compatible cohesive ends are created by PCR and subsequent endonuclease V treatment (as illustrated in Figure 2). The 5′ ends of the PCR primers and the termini of the corresponding PCR products differ from the shown sequences: They lack the first nucleotide (shown in red) and carry deoxyinosine instead of the residue shown in orange. EndoV treatment of the PCR product results in 5′ recessed ends shown in bold letters with yellow background. If the residue highlighted in grey is omitted from the oligonucleotide design, ligation of the insert fragments with linearized plasmid DNA does not reconstitute the restriction enzyme site.