Site-directed mutagenesis is a molecular biology technique that introduces specific changes in DNA sequences to study the structure and biological activity of DNA, RNA, or proteins. It includes various methods for creating mutations, but can be limited by low yields and lengthy protocols. Historically, more random mutation methods were developed before advancements in targeted approaches began in the 1970s, leading to significant applications in research and commercial products.

1. SITE DIRECTED
MUTAGENESIS
By
Sushmitha

2. Mutation
● alteration in the nucleotide sequence of the
genome of an organism, virus, or
extrachromosomal DNA.
Types
1. Germline/ somatic mutation - gametes/ body
cell
2. Chromosomal alteration- chromosome
structure
3. Point mutation- single nucleotide
4. Frameshift mutation - shift in reading frame

3. Site directed mutagenesis
● Molecular biology method- make
specific&intentional changes DNA seq.
● Investigating stuct& bio activity of
DNA,RNA,protein.
● Imp lab techniq for creating DNA libs by
introducing mutations into DNA seq.
● Numerous methods r there- but low cost of
oligonucleotide ,artificial gene syn is used as
alternative method.
Other names
● Site specific
mutagenesis
● Oligonucleotid
e directed
mutagenesis

4. History
● Early attempts at mutation- radiation or chemical mutagens-non
specific , generating random mutation.
● Nucleotides &other chemicals were used to generate localized point
mutation -aminopurine,nitroguanidine,bisulfate.
● 1974-Charles Weissmann- N4-hydroxycytidine - GC to AT.
● 1971-Clyde Hutchison& Marshall Edgell -produce mutants with phage
ϕX174 and restriction nucleases.
● 1978-Hutchison& Michael smith - primer extension method.
● 1993 Michael Smith & Kary B Mullis invent PCR - Nobel prize in
chemistry.

5. Mechanism
● Requires synthesis of short primer.
● Synthetic primer- desired mutations
&complementary to template strand.
● Mutation may be single/multiple base change
,deletion,addition.
● Single strand primer-extended using DNA
polymerase- copies rest of genes.
● Gene copied contain mutated site- introduced
through vector into host cell &cloned.
● Mutants are selected by dna sequencing to check.
● Due to low yield of this method ,several other
methods have been found.

6. Mechanism

7. Other methods
Kunkel’s
method
Whole
plasmid

8. Applications
● Investigative tools-specific mutation in seq allows the function &
property of DNA& protein seq to be investigated.
Eg. Phosphorylation investigated-Particular serine(Phosphate
acceptor) to alanine(Phosphate non acceptor)
● Comercial application : laundry detergents.
● Subtilisin wild type has methionine can be oxidised by bleach-
protein activity 🔻
● methionine 🔄alanine making resistant to oxidation in presence of
bleach-protein activity 🔺
● Oligonucleotide rate🔻 possible to synthesis mutated complete
gene.

9. Disadvantages
● Long amplification protocol - 25cycles(b/w 4 to 8hrs) .
● Low yield of amplified DNA
● Tandem primer insertion at or near the mutated site often
occurs.
● Large insertion and deletion are problematic.
5’ 5’
3’
5’
5’
3’
3’
3’
Template strand
Site to be mutated
Primer
Mutated strand with
tandem primer