Focusing Mutations Within Random Libraries to Distinct Areas: Protein Domain Library Generation by Overlap Extension

Directed evolution is an often used approach toward new proteins with tailor-made properties. It consists of random variation of the coding sequence of a protein followed by an appropriate selection procedure or a suitable type of property read out. In many, if not all cases, it is of significant advantage to constrain the randomly mutagenized DNA sequence to that encoding a particular part of the protein or a distinct domain, and not to mutagenize the entire gene of the target protein. For this purpose, a three-step, polymerase-based method was developed, which is independent of two flanking restriction sites adjacent to the nucleotide sequence supposed to be mutagenized, and named protein library generation by overlap extension (PDLGO).