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