One-tube, two-stage PCR-directed in vitro mutagenesis using megaprimers

▼Mutations very near to the 5′ or 3′ end (up to ∼ 40 bp) of a target sequence are easy to introduce directly by a onestep PCR protocol using mismatching primers. However, it is more difficult in a one-step PCR protocol to introduce mutations large distances from the ends of the DNA template and to obtain a full-length PCR product avoiding random mutations created during the polymerization reaction (Ref. 1). To circumvent such problems, it is necessary to perform the in vitro mutagenesis in a two-stage PCR reaction using a polymerase such as Pwo polymerase which has proofreading activity instead of a conventional Taq polymerase. Two-stage PCR mutagenesis methods based on at least one useful restriction site in close proximity to the target nucleotide sequence (Ref. 2) or methods which use overlap extension in a second PCR step (Ref. 3, 4) always need a set of four primers. Previously described one-step−threestage or one-tube methods often require special equipment or additional working steps. For example, when using biotinylated primers, classical avidin-coated beads cannot be used as they inactivate the Pwo polymerase and so special encapsulated magnetic beads which are more expensive must be used instead (Ref. 5). Other published protocols specify elaborate stages which could be elimated, such as Geneclean purification of megaprimers (Ref. 6), ddNTPblocked DNA as template in the first PCR step (Ref. 7) to prevent wild-type amplification or the toleration of wildtype amplification because of imbalanced primer relation (Ref. 8) or imbalanced cycling parameters which should be optimized for each mutagenesis (Ref. 7). The treatment with Taq polymerase and dATP to add 3′ protruding adenosine to both ends of the product to be cloned in an TA Cloning Kit vector is also unnecessary (Ref. 7).