In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI

Abstract

In this protocol, two oligonucleotides are used to prime DNA synthesis by a high-fidelity polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and have the same starting and ending positions on opposite strands of the plasmid DNA. The entire lengths of both strands of the plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutated plasmid containing staggered nicks on opposite strands. Because of the amount of template DNA used in the amplification reaction, the background of transformed colonies containing wild-type plasmid DNA can be quite high unless steps are taken to enrich for mutant molecules. In this protocol, the products of the linear amplification reaction are treated with the restriction enzyme DpnI, which specifically cleaves fully methylated GMe6ATC sequences. DpnI will therefore digest the bacterially generated DNA used as template for amplification, but it will not digest DNA synthesized during the course of the reaction in vitro. DpnI-resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli cells to antibiotic resistance. Because the method works well with virtually any plasmid of moderate size (<7 kb), it can be used to introduce mutations directly into full-length cDNAs and eliminates the need for subcloning into specialized vectors.