Mutation in Pharmaceutical Biotechnology

PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 1
NANDHA COLLEGE OF PHARMACY
ERODE-52
NAME: DAKSHINESH P
COURSE: B.PHARM
SEMESTER: VI
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PHARMACEUTICAL BIOTECHNOLOGY
MUTATION
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OUTLINE
Definition
Causes
Classification of mutation
Mutants
Isolation of mutants
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DEFINITION:
A mutation is any sudden change in genetic material that may or
may not affect phenotype i.e. the physical expression of a gene.
Mutation can occur due to change in the nucleotide/base sequence
of DNA, due to errors in DNA replication.
• Purines – Adenine and Guanine
• Pyrimidines – Cytosine and Thymine
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CAUSES:
Error in DNA replication or
transcription due to influence of
environmental factors such as with
examples,
Radiation – UV radiation and X-
Rays.
Chemicals- Cigarette smoke,
nitrate and nitrate preservatives,
benzoyl peroxide.
Infectious agents – Human
Papillomavirus, Helicobacter
pylori
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CLASSIFICATION OF MUTATION:
1. Based on the survival of an individual
2. Based on causes of mutation
3. Based on tissue of origin
4. Based on direction of mutation
5. Based on type of trait affected
6. Chromosomal mutation
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1. Based on the survival of an individual:
lethal mutation – causes death of all mutants
Sublethal mutation – causes death of 90% individuals
Sub vital mutation – causes death less than 90%
Vital mutation – do not affect the survival of mutants
Super vital mutation – enhance the survival of mutants.
2. Based on causes of mutation:
Spontaneous mutation – natural without any cause
E.g. UV of sunlight causing mutation in bacteria
Induced mutation - treatment with wither physical or chemical agents.
E.g. X-Ray causing mutation in cereals.
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3. Based on tissue of origin:
Germline mutation are the heritable alterations in the DNA which
occurs in germ cell and it is included in every call of the body. These
mutations are especially significant because they can be transferred to
offspring and every cell in the offspring will have mutations.
Somatic mutations are the modification in DNA that occurs after
conception. It occurs in any other cells of the body except germ cell.
These mutations may have little effect on the organism because they
are restricted to just one cell and its daughter cells. Somatic mutations
cannot be transmitted to next generation.
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4. Based on direction of mutation:
Forward mutation – normal allele to mutant allele
Reverse mutation – mutant allele to normal/wild type allele
5. Type of trait affected:
Visible – affect phenotypic character
Biochemical – affect production of biochemicals and not show
phenotypic character.
6. Chromosomal mutation:
Modification in chromosomal number and structure.
-Deletion, Inversion, Translocation, Nondisjunction, Duplication.
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TYPE OF MUTATION:
Types of mutations can arise due to modification of single pairs
of nucleotides and from addition or deletion of one or two nucleotide
pairs in the coding region of the genes.
Mutation can be differentiated into two major types,
Point mutation
Frameshift mutation
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1. Point Mutation:
A point mutation is a single base substitution that will cause the replacement
of a single base nucleotide with another nucleotide of the genetic material. DNA or
RNA. The individual which is affected by a point mutation is called as point
mutants.
Transitions – In this case there is a replacement of purine base with another
purine are substitution of pyrimidine with another pyrimidine.
Transversions – In this case there is a replacement of purine with pyrimidine or
vice versa.
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Outcomes of point mutation:
i. Silent mutation:
In silent mutation the
mutated codon codes for the same
amino acid. There will be
replacement of single nucleotide
but the new codes specify the same
amino acid, resulting in unmutated
protein
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ii. Missense mutation:
In missense mutation, the
mutated codon codes for the
different amino acid. There will be
single base pair substitution in
DNA that will change a codon for
one amino acid into a codon for
another.
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iii. Nonsense mutation:
In nonsense mutation, there
will be a replacement of single
nucleotide that results in a stop
codon replacing a normal amino
acid codon.
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2. Frameshift Mutation:
A frameshift mutation is insertion or deletion of one or more
nucleotides that alters the reading frame of the base sequence. Deletions
will remove nucleotides whereas will add nucleotides.
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MUTANTS:
Mutants are the individuals
showing mutation.
To detect particular mutant
organism, must be avail of wild
characters.
In bacteria and other haploid
microorganisms the detection
system is simple. Other detection
are complicated.
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ISOLATION OF MUTANTS
1. Replica Platting Technique
2. Resistance Selection Method
3. Substrate Utilization Method
4. Carcinogenicity Test or Ames
method
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1. Replica Plating Technique:
In 1952, Joshua and Esther Lederberg develop replica platting technique.
This technique is used to detect auxotrophic mutants which distinguish between
mutants and wild type strains on the basis of ability to grow in the absence of an
amino acid. This test is used to show the presence of antibiotic resistance in
bacterial cultures prior to exposure of antibiotic.
Steps involved are:
• The mutants are generated by treating a culture with a mutagen i.e.
nitroguanidine.
• Then the plate is inoculated containing complete growth medium and incubated
at proper temperature. Both wild type and mutant survivors will form colonies on
the complete medium. This plate which contain complete medium is called as
master plate.
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• A sterile velvet piece is prepared and is gently pressed on upper surface of
master plate to pick up bacterial cells from each colony.
• As pressed on master plate, the velvet is again gently pressed on replica
plates containing complete medium in one set and lacking only histidine in
other set.
• So, the bacterial cells are transferred in a replica plates in the same position
as in the master plate.
• Then plates are incubated and the replica plates are compared with master
plate for bacterial colony not growing on replica plate.
The Histidine auxotroph (His-) will not grow on replica plates which
do not have leucine. Then, leucine auxotroph are isolated as cultured on
complete medium.
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2. Resistance Selection Method:
This is another approach used for isolation of mutants. Mostly the
wild type cells are not resistant either to antibiotics or bacteriophages.
Thus, it is possible to grow the bacterium in the presence of agent. This
method is applied for separation of mutants resistant to an chemical
compounds that can adjust to agar, phage resistant mutants.
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3. Substrate Utilization Method:
This method is involved in the selection of bacteria. Several
bacteria use only a few primarily carbon sources. The cultures are plated
on a medium which carry on alternate carbon sources. Any colony that
grows on medium can utilize the substrate and are perhaps mutants, and
then these can be isolated. Sugar utilization mutants are also separated
by means of colour is sensitive to pH. This method comprises lactose
sugar as carbon source and complete mixture of amin acids.
Hence, both lactose wild type(Lac+) and lactose mutant (Lac-)
cells can grow and form colonies on EMB agar plate. The Lac+ cells
consumes lactose and secrete acids which will cause decrease in the pH
of medium. It results in staining of colony to dark purple. Whereas,
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Lac- cells are not able to use lactose and utilize some of the amino acids
as carbon source. After usage of amino acid, ammonia is produced that
will increase the pH of medium and decolorise the dye resulting in
white colony.
(Lac+)-----utilise lactose-----Forms acid-----Decrease pH-----Staining
of colony to dark purple colour.
(Lac-)-----utilise amino acid-----Forms ammonia-----Increases pH-----
decolorise the dye-----white colony.
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4. Carcinogenicity or Ames Test:
In 1970s Ames test was developed by Bruce Ames, Professor of Biochemistry at
UC-Berkeley, for evaluating mutagenicity of carcinogens i.e. chemical which is capable of
causing cancer. In Ames test several special strains of Salmonella typhimurium are
involved. Each strain contains a distinct mutation in the operon histidine biosynthesis.
The Ames test follows the following steps:
• The culture of Salmonella histidine auxotroph (His-) are prepared.
• The bacterial cells and test substance i.e. mutagen are mixed in dilute molten top agar
with small amount of histidine in one set, and control with complete medium plus large
amount of histidine.
• The molten mix is poured on the top of minimal agar plates and incubated at 37 degree
Celsius for 2 to 3 days.
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• Till histidine is depleted all the His- cells will grow in the presence of test
mutagens. When histidine is completely depleted only the revertant i.e. the mutants
which have retrieved the original wild type characters will grow on agar plate. The
number of spontaneous revertant is low as compared to number of revertant
induced by the test mutagen which is quite high. The higher the number of
colonies, greater will be mutagenicity.
• Before plating, a mammalian liver extracts is added to the above molten top agar.
• The extract will transfer the carcinogen into electrophilic derivatives which will
soon react with DNA molecule.
It is a natural process which occurs in mammalian system when foreign
substance are metabolized in liver. Bacteria do not have the metabolizing capacity as
liver does, so the liver extract is added in this test just to promote the transformation.
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THANK YOU
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Mutation in Pharmaceutical Biotechnology

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