The principal scheme of the OverFlap strategy for whole plasmid amplification-based randomization of the selected plasmid DNA region.

The methodological approach following circularization is identical to the one described in Fig 1. However, here the plasmid is linearized in a way that the random region of the longer primer does not interact with the template, and an additional stage of asymmetric PCR with only a random region containing a primer is performed to increase the number of sequence variants within the final library. U represents deoxyuridine within the employed primers. The red, black, grey, dotted, and dashed lines represent the randomized region, template DNA, the synthesized chain, DNA synthesis, and template DNA degradation by DpnI restriction endonuclease, respectively.