Two approaches to site-directed mutagenesis. (a) A synthetic DNA segment replaces a fragment removed by a restriction endonuclease. (b) A pair of synthetic and complementary oligonucleotides with a specific sequence change at one position are hybridized to a circular plasmid with a cloned copy of the gene to be altered. The oligonucleotides act as primers for the synthesis of full-length double-stranded DNA (dsDNA) copies of the plasmid that contain the specified sequence change. These plasmid copies are then used to transform cells. (c) Results from an automated sequencer (see Figure 7-12), showing sequences from the wild-type recA gene (top) and from an altered recA gene (recA K72R, bottom) with the triplet (codon) at position 72 changed from AAA to CGC, specifying an Arg (R) instead of a Lys (K) residue. Here, the nucleotide colors reflect the dyes actually used in the method, and thus deviate from the standard nucleotide colors used in other figures.