The Transformer Method
The Transformer Kit uses two oligonucleotide primers that are simultaneously annealed to one strand of a denatured double-stranded template. One primer introduces the desired mutation and the other mutates the unique restriction site in the plasmid, creating a new restriction site or eliminating the site completely. Elongation by T4 DNA polymerase, which lacks strand displacement activity, results in the incorporation of both mutations in the same newly synthesized strand. The DNA is then digested with a restriction enzyme that cuts at the original restriction site. The uncut, mutated DNA will transform E. coli more efficiently than the linear DNA with no mutations.
E. coli BMH 71-18 mutS, which is mismatch repair deficient, is used to propagate the mutated plasmid. Two rounds of DNA digestion and transformation ensure that a very high frequency of transformants carry the mutated plasmid, which nearly always contains both mutations—the desired mutation and the selection mutation (1, 4).
The Transformer Site-Directed Mutagenesis Kit contains enough reagents for 30 mutagenesis reactions, including 10 control reactions. You must supply the selection and mutagenic primers and the vector. The selection primer must contain a mutation that will eliminate a unique restriction site located on the plasmid or will convert the unique restriction site into another unique site. Multiple rounds of mutagenesis may then be performed on the gene of interest without recloning.
Chemically competent BMH 78-18 mutS E. coli cells are available separately for use with the Transformer Mutagenesis Kit. These cells are mismatch-repair deficient.