Exogenous genetic perturbation method nicking mutagenesis A saturation mutagenesis method in which a single-strand break (nick) is introduced in the double-stranded DNA plasmid template using a strand-specific nicking endonuclease. Following the nicking reaction, the nicked strand is degraded by an exonuclease III, generating a single-stranded DNA template. Mutagenic oligonucleotides containing the desired mutations are then annealed to the single-stranded template and extended by a high-fidelity DNA polymerase. The intact strand is then nicked and degraded, and a second primer is used to amplify the mutated plasmid. Nicking mutagenesis can be used to generate user-defined libraries of genetic variants for functional genomics studies.