SITE DIRECTED

MUTAGENESIS
INTRODUCTION

• Site-directed mutagenesis is a molecular biology method that is used to make

specific and intentional changes to the DNA sequence of a gene and any gene products.
Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it
is used for investigating the structure and biological activity of DNA, RNA,
and protein molecules, and for protein engineering.
• Site-directed mutagenesis is one of the most important techniques in laboratory for
introducing a mutation into a DNA sequence. There are numerous methods for achieving
site-directed mutagenesis, but with decreasing costs of oligonucleotide synthesis, artificial
gene synthesis is now occasionally used as an alternative to site-directed mutagenesis.
METHODS OF SDM

• Kunkel’s method
• Cassette mutagenesis
• PCR SDM
Quick change, overlap extension, inverse PCR.
KUNKEL’S METHOD
CASSETTE
MUTAGENISIS
QUICKCHANGE PCR
OVERLAP EXTENSION
INVERSE PCR
APPLICATIONS

• For example, commonly used laundry detergents may contain subtilisin, whose wild-type form
has a methionine that can be oxidized by bleach, significantly reducing the activity the protein in
the process.This methionine may be replaced by alanine or other residues, making it resistant to
oxidation thereby keeping the protein active in the presence of bleach.
• single amino-acid changes by site-directed mutagenesis in proteins can help understand the
importance of post-translational modifications. For instance changing a particular serine
(phosphoacceptor) to an alanine (phospho-non-acceptor) in a substrate protein blocks the
attachement of a phosphate group, thereby allows the phosphorylation to be investigated. This
approach has been used to uncover the phosphorylation of the protein CBP by the
kinase HIPK2