Site-directed mutagenesis is a technique used to generate specific mutations in DNA at predetermined locations. It involves using a synthetic oligonucleotide primer containing the desired mutation to introduce changes into the DNA sequence during in vitro DNA replication or PCR. This allows researchers to study the effects of mutations and engineer proteins with improved or customized properties. Common methods for site-directed mutagenesis include using single or double primers, cassette mutagenesis by replacing DNA fragments, and PCR-based mutagenesis. The technique has various applications in investigating protein function and developing proteins for commercial uses.

1. SITE DIRECTED MUTAGENESIS
Dr. SMT. ARUNIMA KARKUN
(ASST. PROF.)
G.D. RUNGTA COLLEGE OF SCIENCE AND TECHNOLOGY
KOHKA KURUD ROAD BHILAI, 490023
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2.  INTRODUCTION
 HISTORY
 MUTATION
 DIRECTED MUTAGENESIS
 BASIC MECHANISM OF SITE DIRECTED
MUTAGENESIS
 METHOD FOR SITE DIRECTED MUTATIONS
 THE SINGLE PRIMER METHOD
 CASETTEE MUTAGENESIS
 PCR SITE-DIRECTED MUTAGENESIS
 APPLICATION OF SITE DIRECTED MUTAGENESIS
 SUMMARY
 CONCLUSION
 REFERENCES
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3. SITE DIRECTED MUTAGENASIS
• In molecular biology and genetics, mutations are changes in a genomic
sequence.
• Mutations are caused by radiation, viruses, transposes and mutagenic
chemicals, as well as errors that occur during meiosis or DNA replication.
• Site-directed mutagenesis, also called site-specific
mutagenesis or oligonucleotide directed mutagenesis, is a molecular
biology technique often used in bio molecular engineering in which
a mutation is created at a defined site in a DNA molecule.
 Site-directed mutagenesis is the technique for generating amino acid
coding changes in the DNA(gene).
 Site-directed mutagenesis is incorporation of a desired amino acid (of
one's choice) in place of a specific amino acid in a protein or a
polypeptide.
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4. ◦ IN 1791, SETH WRIGTHT first time study in mutations in sheep genome.
◦ IN 1910, MORGEN study in .Mutations in drosophila melangaster.
◦ IN 1927, H.J. MULLER give the CLB method for detection of mutation.
◦ IN 1971, CLYDE HUTCHISON AND MARSHALL EDGELL showed that it
is possible to produce mutants with small fragments of phage φx174 and
restriction nucleases.
◦ IN 1973, CHARLES WEISSMANN using N4-hydroxycytidine which
induces transition of GC to AT .
◦ IN 1978, MICHAEL SMITH site-directed mutagenesis by
using oligonucleotides in a primer extension method with DNA polymerase.
◦ IN 1993, KARY B. MULLIS who invented polymerase chain reaction.
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5.  In molecular biology and genetics, mutations are accidental changes in a genomic
sequence of DNA.
 Mutations can involve large sections of DNA becoming duplicated, usually through genetic
recombination.
 Mutation is may be define as a change in the nucleic sequence (bases) of an organism’s
genetic material (a change in the genetic material of an organism).
 Directed mutagenesis may be define as a change in the nucleic acid sequence (or genetic
material) of an organism at a specific predetermined location.
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6.  Directed mutagenesis may define is a change in the nucleic acid sequence (or
genetic material) of an organism at a specific predetermined location.
 Site-directed mutagenesis is the technique for generating amino acid coding
changes in the DNA (gene). By this approach specific (site-directed) change
(mutagenesis) can be made in the base (or bases) of the gene to produce a desired
enzyme.
 A large amount of experimental procedures have been developed for directed
mutagenesis of cloned genes.
 A synthetic oligonucleotide complimentary to the area of the gene of interest but
has the desired nucleotide change.
 An oligonucleotide is a short piece of DNA usually 10-30 nucleotide long.
 Directed mutagenesis can be done using:
 M13,Plasmid DNA, PCR, Random primers, Degenerate primers,
Nucleotide analogs, DNA shuffling
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Gene in plasmid with target site mutation
Denature the plasmid and anneal the
oligonucleotide.
primers containing the desired mutation
Using the non-strand-displacing action of
PfuTurbo polymerase, extend and incorporate
the mutagenic primers resulting in nicked
circular strands
Digest the methylated, nonmutated parental
DNA template with Dpn I
Transform the circular, nicked dsDNA into
super-competent cells
After transformation the supercompetent cells
repair the nicks in the mutated plasmid
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Fig 1. Basic mechanism of site directed mutagenesis

8. METHOD FOR SITE DIRECTED MUTAGENESIS
 THE SINGLE PRIMER METHOD
 In the technique of oligonucleotide-directed
mutagenesis, the primer is a chemically synthesized
oligonucleotide (7-20 nucleotides long).
 It is complementary to a position of a gene around
the site to be mutated. But it contains mismatch of or
the base to be mutated.
 The starting material is a single-stranded DN A (to
be mutated) carried in an M13, phage vector.
 On mixing this DNA with primer ,the
oligonucleotide hybridizes with the complementary
sequences, except at the point of mismatched
nucleotide.
 Hybridization ( despite a single base mismatch) is
possible by mixing at low temperature with excess of
primer, and in the presence of high salt concentration
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TIONS 8Fig.No.2-single primer method

9.  The addition of 4-deoxyribonucleoside triphosphates and DNA polymerase( usually klenow
fragment of E.Coli DNA polymerase) replication occur.
 The oligonucleotide primer is extended to form a complementary strand of the DNA.
 The ends of the newly synthesized DNA are sealed by the enzyme DNA ligase.
 The double-stranded DNA ( i,e. M phage molecule) containing the mismatched introduced
by nucleotide into E .coli transformation .
 The infected E. Coli cells produce M13 virus particles containing either the original wild
type sequence or the mutant sequence.
 It is expected that half of the phage M13 particles should carry wild type sequence while the
other half mutant sequence (since the DNA replicate semiconservatively).
 The double-stranded DNAs of M13 are isolated.
 Oligonucleotide -directed mutagenesis by using plasmid DNA (instead of M13) is also in
use.
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10.  There are some variations in use in the oligonucleotide directed
mutagenesis,.
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Fig.No.3- Variations in oligonucleotide-directed mutagenesis

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 CASETTEE MUTAGENESIS
 In casettee mutagenesis a, synthetic
double stranded oligonucleotide (a
small DNA fragment i.e., casettee)
containing the requisite/desired mutant
sequence is used.
 Casettee mutagenesis is possible if the
fragment of the gene to be mutated lies
between two restriction enzyme
cleavage sites.
 This intervening sequence can be cut
and replaced by the synthetic
Oligonucleotide (with mutation).
 The plasmid DNA is cut with
restriction enzymes (such as EcoR1
and Hind111).
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Fig.No.4- Casettee mutagenesis

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Fig.No.5- PCR-based mutagenesis

13.  The PCR-based mutagenesis technique commonly employed is depicted in First the target DNA
(gene) is cloned on to a plasmid vector and distributed in to two reaction tubes.
 To each tube are added two primers ( oligonucleotides synthesized by using PCR).
 One primer ( A in tube1 and C in tube 2) is complementary to a region in one strand of the cloned
gene except for one nucleotide mismatch( i.e. the one targeted for a change).
 The other primer (B in tube 1 and D in tube2 ) is fully complementary of a sequence in the Other
strand with in or adjacent to the cloned gene.
 The placement of primers for hybridization ( with the DNA strands) in each tube is done in
opposite direction.
 The PCR technique is carried out for amplification of the DNA molecule.
 The products of PCR in the two reaction tubes are mixed.
 The DNA molecules undergo denaturation and renaturation.
 A Strand from one reaction tube (strand A) hybridizes with its complementary strand from other
reaction tube (strand C). 13
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14.  Site-directed mutagenesis is used to generate mutations that may
produce rationally designed protein that has improved or special properties
 Investigative tools - specific mutations in DNA allow the function and properties
of a DNA sequence or a protein to be investigated in a
rational approach.
 Commercial applications - proteins may be engineered to produce proteins that
 are tailored for a specific application.
 Example, commonly-used laundry detergents may contain subtilise in whose
wild-type form has a methionine that can be oxidized by bleach, inactivating the
protein in the process.
 This methionine may be replaced by alanine, thereby making the protein active in
the presence of bleach.
 Site-directed mutagenesis has been widely used in the study of protein functions.
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15. • Any amino acid in a protein can be selectively replaced with
another naturally occurring amino acid
• The replacements are made at the genetic level by modifying
the codon to incorporate the new amino acid
• Characterizing the mutant enzyme that is obtained will
provide information on the role of the amino acid that has
been replaced
• The only unequivocal result from mutagenesis studies is
when the mutation has no effect on the enzyme’s function.
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16. • Mutations are caused by radiation, viruses, transposes and mutagenic
chemicals, as well as errors that occur during meiosis or DNA
replication.
• Site-directed mutagenesis, also called site-specific mutagenesis or oligo
nucleotide-directed mutagenesis, is a molecular biology technique often
used in bio molecular engineering in which a mutation is created at a
defined site in a DNA molecule.
• Directed mutagenesis may define is a change in the nucleic acid
sequence (or genetic material) of an organism at a specific
predetermined location.
 Mutation, a change in the nucleic sequence (bases) of an organism’s
genetic material (a change in the genetic material of an organism).
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