Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA sequence of interest. It requires relatively little preparation compared with other mutagenesis methods and does not require the use of restriction enzymes. Because of its versatility, the method has become widely used. Unlike methods of random mutagenesis, directed mutagenesis requires that the researcher already have a specific mutation in mind to implement. Traditional overlap extension PCR mutagenesis protocols remain limited in several critical ways, especially when it comes to generating insertions and deletions. For example, traditional protocols require that all sequence alterations be embedded within the primer itself, which makes it difficult to make insertions >30 nt. This protocol describes an overlap extension PCR mutagenesis method that is more versatile than its predecessors. Using this method, one can essentially make insertions and deletions of any size at any position within a given DNA sequence. To generate an insertion mutation, first prepare an insertion fragment and two flanking fragments by PCR. In the secondary PCR, the insertion fragment is recombined with two flanking fragments derived from the original template. This method can also be used to generate deletions, which is discussed in the latter part of the protocol.