Roughly based on Chapter 11 Biotechnology: Principles and Processes and Chapter 12 Biotechnology and its Applications of Class 12 NCERT for final brush-up before the exams

1. BIOLOGY
Chapter: Biotechnology and its
Applications

2. • What is biotechnology?
Ans.: Biotechnology is defined as the broad area
of biology which uses both the technology and the
application of living organisms and their components
to develop, modify and produce useful products for
human welfare.
• What is the purpose of biotechnology?
Ans.: The purpose of biotechnology is to help humans
to get to fulfill the growing population and to get
better result with lesser efforts and resources.

3. Microbe- mediated processes (making
curd, bread, wine etc.)
In vitro fertilization (“test- tube baby
programme”)
Synthesis and using of a gene
Preparation of vaccine
Correction of defective gene (gene
therapy)
Production of high quality crops in great
quantity with minimum resources and
wastage
Biotechnology deals
with

4. Overview of Biotech Processes
1. Isolation of pure DNA
2. Cutting of DNA
3. Separation and Isolation of DNA fragments (AGE)
4. Elution of DNA fragments from gel
5. Introduction of desired DNA fragments into vector
6. Transfer of recombinant in to a competent host
7. Selection of recombinants
8. Colonies of recombinants
9. Obtaining gene product

5. Main component
of cell wall
Enzyme needed Image
PLANT CELL Cellulose Cellulase
FUNGUS
CELL
Chitin Chitinase
BACTERIAL
CELL
Peptidoglycan Lysozyme
1. ISOLATION OF GENETIC MATERIAL
i. Cell Wall:

6. ii. Cell Membrane:
• Composed of: LIPID BILAYER
• Enzyme required: LIPASE
iii. Cytoplasm:
PROTEIN DNA RNA
CHILLED
ETHANOL
LONG THREADS OF DNA
STRAND (REQUIRED)
The process is called SPOOLING
RNAse
PROTEASE

7. Restriction Enzyme
• Main component for this: Restriction Enzyme
• Also known as Molecular Scissors
2. CUTTING
RESTRICTION ENDONUCLEASE RESTRICTION EXONUCLEASE
BREAKS PHOSPHODIESTER
BOND AND BREAKS THE DNA
MOLECULE FROM THE
MIDDLE PARTS OF IT
BREAKS PHOSPHODIESTER
BOND AND BREAKS THE DNA
MOLECULE FROM THE ENDS
ONLY

8. Palindromic sequence
The sequence of nitrogen bases which read the same for
both the complementary strands of DNA when read from
the same direction
•Restriction enzymes cut at
specific parts or specific
palindromic sequences
•It forms identical sticky ends at
both the foreign and vector
DNA
•Both vector and foreign DNA
are cut by the same restriction
enzyme so that they can attach
to each other
Recognition sequence
for restriction enzyme
EcoRI

9. • Most used and common technique: Gel Electrophoresis
• The gel is made of: Agarose
• Dye used: Ethidium Bromide
• Done in presence of UV radiation
• Process of extracting DNA from
gel: Elution
3. SEPARATION AND EXTRACTION OF DESIRED DNA
CATHODE END
ANODE END
-
+
PRINCIPLE OF AGE:
DISTANCE TRAVELLED IS INVERSELY
PROPORTIONAL TO THE SIZE OF
THE DNA FRAGMENT
Both the dye and UV
radiation are Carcinogenic.
So precautions need to be
taken.

10. 4. FORMATION OF RECOMBINANT DNA (rDNA)
Overview of the process hoe rDNA
is formed
A scaled up image of the exact
region

11. PLASMID VECTOR:
COMPONENTS OF PLASMID VECTOR:
1. Origin of replication-
1.1. starting point of replication
1.2. controls the number of copies of DNA
2. Selectable markers-
3. Cloning site- the site where foreign DNA is introduced
The vectors are usually smaller in
size so that the introduction into
host body is faster

12. An example of a plasmid vector: pBR322
NAMING OF PLASMID VECTOR-
p- plasmid
B- Bolivar
R- Rodriguez
322- order of synthesis
pBR322 plasmid
Recombinant Plasmid
Introduction to host cell and
grown in-vitro
Non-Recombinant Plasmid
No growth Growth

13. 5. AMPLIFICATION (POLYMERASE CHAIN REACTION)
•In vitro amplification of desired DNA sequence
•Discovered by- Kary Mullis
•Done at a high temperature (90˚-100˚ C) so thermostable enzyme is needed
•Name of enzyme required- Taq polymerase which is found in Thermus
aquaticus
Procedure of PCR:
(50˚ -60˚ C)
(90˚ -95˚ C) (72˚ C)

14. Application of PCR:
1. Diagnosis of genetic diseases
2. Generic fingerprints (forensic studies, paternity test etc..)
3. Detection and diagnosis of infectious diseases
4. Detection of infection in the environment
5. Personalized medicine
VNTR (Variable Number Tandem Repeats) : these are mini satellite DNA i.e. a repetition
of 35-80bp sequences to a size of 2kbp which are found repetitively in a DNA which are
unique to individuals.
By matching the location of the sequence paternity test and forensic studies are dome.
A simple formula can be used to find out the no of DNA formed after each PCR.
Total no of DNA formed= 2ˆn (where n= no of cycles in PCR)
Example:
Q1. if 5 cycles of PCR done how many DNA are formed?
Ans.- 2ˆ5=32
Q2. if we have 16 DNA how many cycles of PCR done?
Ans .- 2ˆn=16 so n= 4

15. •To avoid this problem the host cell is treated with dipositive Calcium cations ( )
•This is done to increase the Pore Size of the Cell membrane
•Increased pore size help the entry of the rDNA into the host cell
6. INSERTION OF RDNA INTO HOST CELL
HOST CELL
LIPID BILAYER PRESENT
OUTSIDE
HYDROPHOBIC IN
NATURE
rDNA
A TYPE OF DNA (NEGATIVELY
CHARGED)
HYDROPHILIC IN
NATURE
•COMPETENCY:
Since both are of opposite nature, repulsion is observed
This process of treating the host cell in a way so that it is competent for the entry of
DNA in it is called competency

16. Insertion o rDNA into host cell
Bacterial cell
done by
Heat shock
method
Plant cell
done by
Biolistic/gene
gun
Animal cell
done by
Microinjection

17. BIOREACTOR
These are special types of devices specially designed to perform reactions and generate
products in a large scale in industries. Mainly large scale fermentation takes place in these.
Bioreactors included in the syllabus:
Sparged bioreactor

18. EXTRACTION OF PRODUCT
The process by which extraction of product is done, is called Downstream Processing.
Downstream processing refers to the process of purification and separation of products of
recombinant DNA technology. Suitable preservatives are added to the product .

19. APPLICATIONS OF BIOTECHNOLOGY
1. In plants
i. Pest resistant plants
ii. Biofortification
iii. Abiotic- Stress resistive plants
2. In medicine
i. Genetically engineered insulin
iii. Gene therapy
Transgenic
plants
Eg. Superbug
4. Other genetically
modified organisms
3. In Animals
Transgenic animals
ii. Molecular diagnostics

20. 1. APPLICATION IN PLANTS
i. PEST RESISTANT PLANT:
Plants are modified in a way that no pesticide is needed to protect them from pests.
Examples:
Bollworm infected cotton
Healthy cotton pod
•Bt Plants:
a. Infecting organisms: cotton bollworms, corn borers etc..
b. Main infecting agent in modified plants: Bt toxin
c. Toxin producing organism: some strains of Bacillus thuringiensis
d. Gene responsible: cry gene
e. Examples of Bt plants: Bt cotton, Bt corn, rice, tomato etc..
f. Course of action:
cry gene from Bacillus
thuringiensis
Makes perforations
in the gut lining
Insect dies
Inserted in
plants
Production of
inactive CRY protein
in plant
It comes in contact with
the alkaline gut of insect Insect eats the plant
Activation of
CRY protein
Active protein attaches to
mid gut lining

21. •Pest resistant plants using RNAi: (Only for Eukaryotes)
a. Infecting organisms: Nematode parasites (eg. Meloidegyne incognitia)
b. Protective process: RNA interference (RNAi) (Gene silencing)
c. Method: Nematode specific gene introduced into plant body
d. Vector for introduction of gene: Agrobacterium tumefaciens vector (Ti plasmid)
e. Example of plant : Tobacco
f. Course of action:
Infected roots
Central Dogma
DNA (ds)
PROTEIN
RNA (ss)
Transcription
Translation
dsDNA introduced
into plant root cell
Insect
dies
Produces
dsRNA
Dicer enzyme
breaks dsRNA into
small fragments
Unzipping of
the small RNAs
by RISC factor in
worm body
Enters worm body
when consumes
plant root
Unzipped short
ssRNA fragments
bind with worm
ssRNA
Forms incomplete
dsRNA in the worm
Translation and thus
protein synthesis is
stopped

22. ii. Biofortification:
Plants are modified in a way that their nutritional value increases.
Examples:
•Golden rice:
The rice is genetically modified to produce beta carotene
which gets converted to vitamin A when metabolized by
human body.
When consumed regularly this can improve eyesight and
skin health.
•Other examples can be: biofortified sweet
potato, tomato etc..
Normal rice
Golden rice
ii. Abiotic- Stress resistive plants:
Plants are modified in a way that they can endure harsh climatic condition, drought,
submergence, flood, etc.. Post harvest loss is also protected.
These plants can endure
a) Oxidative stress,
b) Salt stress depending on the modification made on them.
Examples: Mainly observed for rice grown in extreme conditions

23. 2. APPLICATION IN MEDICINE
i. GENETICALLY ENGINEERED INSULIN:
About Insulin:
•Insulin (51 Amino acids) is made of two short
polypeptide chains, A chain (31 Amino acids) and B chain
(20 Amino acids) joined by disulphide bonds
•In mammals insulin is produced is a pro-hormone which
is inactive. This inactive form contains an extra C peptide
chain which gets removed when matured
About genetically engineered insulin:
•1st produced in 1983 by Eli Lilly, an American company
•2 DNA sequences corresponding to A and B chains of human insulin were separately
introduced in E. coli plasmid. Separately produced A and B chain are later extracted and
joined by creating disulphide bonds to form human insulin
ii. MOLECULAR DIAGNOSTICS:
•Early disease detection done by Polymerase Chain Reaction, Enzyme Linked Immuno-
Sorbent Assay (ELISA), Recombinant DNA Technology.
•Detection of mutated genes in suspected cancer patients can be done too
•PCR is routinely used to check for HIV in suspected AIDS patients

24. iii. GENE THERAPY:
Gene therapy is a collection of methods that allows correction of a gene defect that has
been diagnosed in a child/ embryo.
•1st given in 1990.
•The 1st recipient was a 4 year old girl suffering from
Adenosine Deaminase (ADA) deficiency.
Adenosine deaminase
enzyme
Important for lymphocyte
development
•Due to lack of Adenosine deaminase the lymphocyte production is also reduced. So
immunity is also compromised. This ultimately caused Severe Combined Immuno
Deficiency (SCID).
•Treatments in Gene Therapy:
a) Enzyme replacement therapy- ADA injection every month
b) Bone marrow transplant- for permanent cure
c) Embryonic level treatment
•Steps in gene therapy:
a) Lymphocyte from patient grown in culture media
b) Functional ADA cDNA using retroviral vector is introduced in the lymphocytes of
patient
c) Modified ADA cDNA is returned into patient

25. 3. APPLICATION IN ANIMALS
TRANSGENIC ANIMALS:
Animals that have their DNA manipulated
to possess and express an extra (foreign)
gene are known as transgenic animals.
Examples:
Transgenic mice, pigs, rabbits, cows,
sheep, fish etc. are also found.
Benefits:
1. Study of normal physiology and
development
2. Study of disease models
3. Vaccine safety and drug testing
4. Production of biological products
5. Possible source for human organ
harvesting

26. 4. OTHER TYPES OF GMO: SUPERBUG
•It is a special type of genetically modified organism
•Pseudomonas putida is a bacteria which is used to treat
oil spills in large water bodies from oil tankers. It is used
to protect nature.
•It is used to degrade hydrocarbons found in petroleum
wastes
•It contains 4 types of plasmids which can degrade 4
different types of hydrocarbons
•Prof. Ananda Mohan Chakraborty received Nobel prize in
1980 in chemistry along with Gilbert and Paul Berg.
When a particular feature of a microorganism is altered by the entry of
an foreign gene, those microorganisms are called transgenic organisms.
Example: Pseudomonas putida (superbug)

27. ETHICAL ISSUES
• DBT (Department of Biotechnology) developed the Recombinant DNA Safety
Guidelines (1990) to enforce the Environment Protection Act, 1986 in India.
• The GEAC (Genetic Engineering approval Community) under the Ministry of
Environment and Forests ensures overall safety for all types of GMOs, experimental
use and commercial use of transgenic plants, animals, GM crops, medical products
and grants clearance in accordance with regulations.
BIOPIRACY AND BIOPATENT
• Biopiracy: Biopiracy is the use of a country's natural resources for research
and commercial purposes without a contract or permission by obtaining a
patent. Ex: basmati rice, neem etc.
• Biopatent: A Biopatent is a legal permit that allows an individual or
organization to use an organism, organic product or biological method for
commercial purposes.