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What Is a Mutation?
Genetic information is encoded
by
the
sequence
of
the
nucleotide bases in DNA of the
gene. The four nucleotides are:
adenine (A), thymine (T), guanine
(G), and cytosine (C), a mutation
is a change in the order of these
nucleotides.
A change in the order can cause
the gene to encode for wrong
proteins and inhibit the function
of the gene or cause the gene to
be virtually inactive.
Random mutagenesis
-> based on the process of natural evolution
- NO structural information required
-NO understanding of the mechanism
required
General Procedure:
Generation of genetic diversity
 Random mutagenesis
Identification of successful variants
 Screening and seletion
Site-directed Mutagenesis
Site Directed Mutagenesis is a molecular biology
technique in which a mutation is created at a
defined site in a DNA molecule known as a
plasmid. Wild-type gene sequence must be known.
Or
Site Directed Mutagenesis is a powerful technique
where site specific changes in DNA sequence are
produced in vitro-for instance to change an amino
acid residue into another by changing the codon
sequence within the gene sequence.
Wild type
CAG
CAG
GTC
GTC
(5)
(1)
CAG
translation
Val
Wild type protein
+ primer
(2)
Mutant
CGG
GCC
CAG
primer
GCC
translation
(6)
+ polymerase
(3)
Thr
CAG
(4)
Mutant protein
GCC
replication
Smith (1993)
Only one amino acid changed
Val → Thr
Genetics
Mutant phenotype
Mutant allele
DNA sequence
Protein sequence
Why Site directed mutagenesis
-> site-directed mutagenesis
-> point mutations in particular known area
result -> library of wild-type and mutated DNA (sitespecific)
not really a library -> just 2 species
-> random mutagenesis
-> point mutations in all areas within DNA of interest
result -> library of wild-type and mutated DNA
(random)
a real library -> many variants -> screening
!!!
if methods efficient -> mostly mutated DNA
INVENTION
Site Directed Mutagenesis using oligonucleotide
was first described in 1978 by Michael Smith &
shared Nobel Prize in chemistry in October 1993
with Kary B. Mullis who developed the PCR
technique.
 Site –directed mutagenesis
Requirements:
-> Knowledge of sequence and preferable Structure
(active site,….)
-> Understanding of mechanism
(knowledge about structure – function relationship)
Site Directed Mutagenesis
1. Cassette mutagenesis
2. Oligonucleotide directed mutagenesis
 Using M13 DNA OR
 Using Plasmid DNA
3. PCR amplified Oligonucleotide directed
mutagenesis
4. Random mutagenesis
 With Degenerate Oligonucleotide primers
or
 Using Nucleotide Analogues
CASSETTE MUTAGENESIS
Cleavage by a Restriction
Enzyme (RE) at a particular site
in the plasmid.
Ligation of an Oligonucleotide
containing Mutation in the gene
of interest to the plasmid.
RE that cuts at the plasmid
and Oligonucleotide is same,
permitting sticky ends of the
plasmid & inserts to ligate to
one another.
Site-directed mutagenesis methods –
Oligonucleotide - directed method
Oligonucleotides are…
 Synthetic single-stranded fragments of DNA used for
priming the mutated clones.
 In order to work, the primers must meet the
following criteria:



-must contain desired mutation.
-mutation should be in the middle of the primer.
-the GC content should be at a minimum of 40%
and should terminate in one or more of C or G
bases.
Oligonucleotide directed
mutagenesis
Basic Methods
1. Base pair substitution: Change
of one nucleotide (A-> C)
2. Insertion: Gaining one
additional nucleotide
3. Deletion: Loss of one nucleotide
Deletion Mutagenesis
Oligonucleotide directed
mutagenesis with M13 DNA:
1. Synthesize an Oligonucleotide
containing the changed sequence.
Ex.---ATT---Wild type sequence
(Codon for ILe)
---CTT---Desired Change (Leu)
---GAA---Mutagenic
Oligonucleotide (MO)
2.Hybridize MO ss form of
gene cloned into M13.
3.Synthesize second strand of
DNA with KLENOW fragment
&dNTPs.
4.Seal nick in new strand
with T4 DNA ligase.
5.Introduce into E. Coli.
6.ss + phage isolated from
plaques
&
screened
by
hybridization.
Oligonucleotide
Directed
Mutagenesis
Using Plasmids
Increases the chances of
obtaining
the
desired
mutation in the target
gene
Co selection technique
Antibiotic resistance gene
restored and a second one
eliminated
Approx 90% of Ampr and
Tets clones have target
mutated
in
directed
fashion
Primer design for PCR-Amplified
Oligonucleotide-directed
mutagenesis
Site-directed mutagenesis
methods -PCR based
Perform
two
separate PCR rx.
With two sets of
primers.
Each primer set
introduces
the
desired nucleotide
change
but
produces
linear
PCR products with
different ends.
Denaturation,
mixing & ligation
of PCR products
produces mutated
circular DNA that
can be introduced
into E. Coli by
transformation.
Random Mutagenesis Using Degenerate
Oligonucleotide Primers
Make primers at sites
to be mutated which
are degenerate at one
or more sites
Random changes in
nucleotide sequences
of the primer are
introduced
during
chemical synthesis of
a pool of degenerate
oligonucleotides.
Must express mutant
genes & screen gene
products to find the
improved version of
the PROTEIN.
Random
Mutagenesis With
Nucleotide Analogue
Cut clone to side of target gene
Digest with ExoIII
Synthesize with DNAP I, dNTPs
and a dNTP analog.Ex.N4-hydroxy
cytosine is an analog of cytosine.
Presence in DNA causes an A-T
base pair transition mutation
when the DNA is replicated by the
host. Ex.
---AAA---(Lys) to ---GAA---(Glu)
---TTT---…………---CTT--Analog causes replication errors
when transformed into E. coli
Uses of Site Directed Mutagenesis
1.Site Directed Mutagenesis is also used to ‘engineer’
commercially important Proteins for many different purposes,
for example
•Improve stability
•Change specificity
•Reduce toxicity
2.It can be used to produce proteins which can ‘improve’
laundry detergents
SUBTILISIN, a bacterial serine-protease that can breakdown
most proteins which soil clothing & a major additive to modern
‘BIO’ detergents. Before it became commercially useful, the
recombinant protein was improved in order that it remained
active in the presence of Bleach. Bleach caused the oxidation of
a methionine residue (position 22) & the enzyme lost 90% of its
activity. By replacing this amino acid with alanine, the enzyme
was no longer sensitive to oxidation & found best in terms of
ACTIVITY &STABILITY.
 3. Site Directed Mutagenesis enabled new
approaches to drug designing – particularly in
order to improve FUNCTION.
 4.Random Mutagenesisis is used to construct
large &diverse clone libraries of mutated DNA
fragments.
FUTURE ASPECTS
Attempts are being made to produce biotechnically a
mutated haemoglobin which may give us a new means of
replacing blood.
By mutating proteins in the immune system researchers
have come a long way towards constructing antibodies that
can neutralize cancer cells.
Future also holds possibilities of gene therapy, curing
hereditary diseases by specifically correcting mutated code
words in the genetic material.
Site directed mutagenesis of plant proteins is opening up
the possibility of producing of producing crops that can make
more efficient use of atmospheric carbon dioxide during
photosynthesis.
Conclusion:
• any amino acid in a protein can be selectively
replaced with another 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
REFERENCES:
 Old R.W. and Primrose S.B. (2001), Principles Of Gene
Manipulation, Blackwell Scientific Publications, Page (132138)
 Brown T.A. (2001) Gene cloning and DNA analysis – An
Introduction Blackwell Publication Page – (236 – 241)
 Watson J.D., Gilman M, Zoller M and Witkowski(1992)
Recombinant DNA, Scientific American Books, Page (191207)
 En. Wikipedia. Org/wiki /site directed mutagenesis 32K
 WWW. Che.Utoledo. Edu / nadarajah / WebPages /
mutagene. Htm.14K
 Glick, Molecular Biotechnology Page (171 – 183)