Saturation mutagenesis by cassette insertion introduces a library of site-specific changes into a specific DNA sequence within a target gene and is especially useful for analyzing the effect of specific residues on the structure and function of a protein. In this protocol, a set of 11 universal oligodeoxyribonucleo-tide cassettes is used to generate mutations. The major advantage of this method is that a single set of mutagenic codon cassettes can be used to insert codons encoding all possible amino acids at any predetermined site within a gene. Each of the 11 cassettes contains two recognition sequences for SapI, a restriction enzyme that cleaves outside of its recognition sequence. The recognition sequences for SapI are arranged in opposite orientations and are separated by a central spacer. At the end of each cassette is a 3-bp direct repeat, positioned such that the sites of SapI cleavage bracket each repeat. Cleavage by SapI will result in the generation of three base-cohesive single-stranded ends on the end of the cassette. These three base-cohesive single-stranded ends can then be ligated together to regenerate the original 3-bp direct repeat, while excising the central spacer. It is this 3-bp repeat sequence that is ultimately incorporated into the template. By substituting the 3-bp direct repeats in the universal cassette with other sequences, one can essentially generate all possible amino acid substitutions.