Two-Stage Polymerase Chain Reaction Protocol Allowing Introduction of Multiple Mutations, Deletions, and Insertions, Using QuikChangeTM Site-Directed

The invention of polymerase chain reaction (PCR) (1 ,2 ) provided a powerful tool to modify DNA sequences in genetic engineering. With numerous mutagenesis methods available, such as traditional sequential PCR (3 ), “megaprimer PCR”(4 –7 ), marker-coupled PCR (8 ), and so on, introducing changes to DNA sequences has become less tedious and more efficient. Recently, marketed site-directed mutagenesis (SDM) kits, such as Transformer™ Site-Directed Mutagenesis Kit (Clontech, San Francisco, CA), and Altered Site� II in vitro Site-Directed Mutagenesis Systems (Promega, Madison, WI), even eliminate the necessity of subcloning the amplified fragment.