Genetic engineering

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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