Cassette mutagenesis is a type of site-directed mutagenesis that uses a short, double-stranded oligonucleotide sequence (gene cassette) to replace a fragment of target DNA. It uses complementary restriction enzyme digest ends on the target DNA and gene cassette to achieve specificity. It is different from methods that use single oligonucleotide in that a single gene cassette can contain multiple mutations. Unlike many site directed mutagenesis methods, cassette mutagenesis also does not involve primer extension by DNA polymerase.