A plasmid containing a target gene YFG (black segment) is cleaved with restriction enzymes XbaI and BglII, each of which has unique restriction site in the entire plasmid. The reaction mixture is separated by agarose gel electrophoresis. Two ss oligonucleotides are synthesized by automated DNA synthesis. The sequences of the oligonucleotides are complementary to each other and differ from wild-type sequence at only a single position (black stripe) containing the desired changes. The oligonucleotides are mixed that helps in hybridization of the two strands by complementation. The ends of the duplex fragment are single-stranded, sticky ends that join with XbaI and BglII sites. The DNA cassette is mixed with the isolated fragment and two molecules are covalently joined by action of T4 DNA ligase. The ligated DNA is transformed into E. coli, and drug-resistant colonies are selected. Plasmid DNA is prepared from individual bacterial colonies. Since the two linear fragments themselves cannot transform E. coli, all colonies contain plasmids with the mutant sequence.

3-6.2.3 PCR based site Directed Mutagenesis

The limitation of restriction sites in cassette mutagenesis may be overcome using polymerase chain reaction with specific oligonucleotide "primers", such that a larger fragment may be generated covering two convenient restriction sites. The exponential amplification in PCR produces a fragment having the desired mutation in sufficient quantity to be isolated from original, unmutated plasmid by gel electrophoresis. There are many variations of the same technique. The simplest method results the mutation site towards one of the ends of the fragment whereby one of two oligonucleotides used for generating the fragment contains the mutation. This involves a single step PCR, but still has the problem of requiring a suitable restriction site near the mutation site unless a very long primer is used. Other variations therefore employ three or four oligionucleotides, two may be non-mutagenic oligonucleotides that cover two convenient restriction sites and generate a fragment that can be digested and ligated. Mutagenic oligonucleotides may be complementary to a location within that fragment well away from any convenient restriction site. These methods require multiple steps of PCR so that the final fragment to be ligated can contain the desired mutation.