INTRODUCTION, HISTORY, MUTATION, DIRECTED MUTAGENESIS,BASIC MECHANISM OF SITE DIRECTED MUTAGENESIS,METHOD FOR SITE DIRECTED MUTATIONS,THE SINGLE PRIMER METHOD, CASETTEE MUTAGENESIS, PCR-SITED DIRECTED MUTAGENESIS, APPLICATION OF SITE DIRECTED MUTAGENESIS.

1. Site Directed Mutagenesis
BY - VIPIN SHUKLA
Himt College of biotechnology
Greater Noida
By – Vipin Kumar Shukla

2. Contents:
• 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

3. Introduction
• In molecular biology and genetics, mutations are the changes in
genomic sequence.
• Mutations are caused by radiations, viruses and transposes and
mutagenic chemicals as well as errors that occur during Meiosis
or DNA replication.
• Site- Directed mutagenesis is also called as site-specific
mutagenesis or oligonucleotide-directed mutagenesis, it is used
for investigating the structure and biological activity of DNA
RNA and Protein molecules.
• 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.

4. HISTORY
Of Site-Directed Mutagenesis
• IN 1791, SETH WRIGHT 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.

5. Contd….
• 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 oligo-nucleotide complimentary to the area of the
gene of interest but has the desired nucleotide change.
• An oligo-nucleotide 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

6. Steps involved in site directed mutagenesis:
• 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

7. Methods:
• METHOD FOR SITE DIRECTED MUTAGENESIS:
THE SINGLE PRIMER METHOD:
• In the technique of oligo-nucleotide-directed mutagenesis, the
primer is a chemically synthesized oligo-nucleotide (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 DNA(to be mutated)
carried in an M13, phage vector.
• On mixing this DNA with primer ,the oligo-nucleotide
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

8. Methods:
• fragment of E. Coli DNA polymerase) replication occur.
• The oligo-nucleotide 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 ( ie. 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 semi-conservatively).
• The double-stranded DNAs of M13 are isolated.
• Oligo-nucleotide -directed mutagenesis by using plasmid DNA
(instead of M13) is also in use.

9. Cassette 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).

10. PCR Based Mutagenesis:
• 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.

11. Applicatons:
• 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.

12. Applications:
• 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.

13. Summary:
• 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).