Certificate

This is to certify that this biology project on the topic

Application of biotechnology in the field of Medicine has
been successfully completed by Prasoon Singh of class 12th
A4 studying in Rani Laxmi Bai Memorial School,
Lucknow under the guidance of biology teacher Shikha
Tripathi (subject teacher) during the academic year 2023-
2024 as per the guidelines issued by Central Board of
Secondary Education (CBSE).

Internal Examiner External Examiner

 Acknowledgement

I would like to extend my sincere and heartfelt obligation

towards all those who have helped me in making this project
without their active guidance, help, co-operation and
encouragement; I would not have been able to make this
project on time.
I am extremely thankful and pay my gratitude towards
my Biology teacher for her valuable guidance and support
for the completion of this project.
I also acknowledge with a deep sense of gratitude towards
my parents and friends for the valuable suggestions given
for this project.
 Introduction
Biotechnology is technology based on biology, especially
when used in agriculture, food science and medicine.

The term brings to mind many different things. Some

think of developing new types of animals. Other dream of
almost unlimited
sources of human
therapeutic drugs.
Still others envision
the possibility of
growing crops that
are more nutritious
and naturally pest-
resistant to feed a
rapidly growing
world population. This question elicits almost as many
questions as there are people to whom the questions can be
posed.

This topic deals with basic principles of biotechnology, the

components to the process of gene cloning such DNA
manipulative enzymes and vectors which transport the
 desired gene into host
cell. Latter part of the
chapters turns our
focus to PCR process
and applications along
with obtaining the
desired product on large scale using bioreactors.

In the purest form, the term biotechnology refers to the use

of living organisms or their products to modify human
health and the human environment. However, it is used in a
restricted sense today, to refer to those processes which use
genetically modified organisms to achieve the same on a
larger scale. Further, many other processes/techniques are
also included under biotechnology.
The European Federation of Biotechnology (EFB) has given
a definition of biotechnology that encompasses both
traditional view and modern molecular biotechnology. The
definition given by EFB is as follows

The integration of natural science and organisms,

cells, parts thereof, and molecular analogues for
products and services
Principles of Biotechnology
The two core techniques that enabled us to combine the
genetic elements of two or more living cells or that enabled
birth of modern biotechnology are:

(i). Genetic Engineering: Techniques to alter the

chemistry of genetic material (DNA and RNA), to
introduce these into host organisms and thus change the
phenotype of the host
organism. The recombinant
DNA thus created is called
rDNA or chimeric DNA
which has properties of DNA
from multiple sources.

(ii). Bioprocess Engineering: Maintenance of

sterile(microbial contamination-free) ambience in
chemical-engineering processes to
enable growth of only the desired
microbe/eukaryotic cell in large
quantities for the manufacture of
biotechnological products like
antibiotics, vaccines, enzymes,etc.
In these techniques, functional
lengths of DNA can be taken from one organism and
placed into the cells of another organism.

There are three basic steps in genetically modifying

organisms that are listed below:
i. Identification of DNA with desirable genes.
ii. Introduction of the identified DNA into the host.
iii. Maintenance of introduced DNA in the host and
transfer of the DNA to its progeny.
 Recombinant DNA
Technology
Recombinant DNA technology alters the phenotype of an
organism (host) through a genetically altered vector. This
cloning vector is introduced and integrated into the genome
of the organism. So, basically, the process involves the
introduction of a foreign piece of DNA into the genome
which contains our gene of interest. The gene which is
introduced is the recombinant gene and the technique is
called the recombinant DNA technology. Here we will learn
about key tools of recombinant DNA technology.

Tools required to accomplish genetic engineering

includes:
1. DNA manipulative enzymes: Inserting the
desired gene into the genome of the host is not as
easy as it sounds. It involves the selection of the
desired gene for administration into the host
followed by a selection of the perfect vector with
which the gene has to be integrated and
recombinant DNA formed. This recombinant
DNA then has to be introduced into the host. And
 at last, it has to be maintained in the host and
carried forward to the offspring. Recombinant
DNA technology can be complete and achieved with
the help of some elemental tools. The different tools
used for the purpose are discussed below:

 Restriction Enzymes: The restriction enzymes – help

to cut, the polymerases- help to synthesize and the

ligases- help to bind.

The restriction enzymes used in recombinant DNA

technology play a major role in determining the location at
which the desired gene is inserted into the vector genome.
They are two types, namely endonucleases and
exonucleases. The endonucleases cut within the DNA strand
whereas the exonucleases cut the nucleotides from the ends
of the DNA strands. The restriction endonucleases are
sequence-specific which is usually palindrome sequences
and cut the DNA at specific points. They scrutinize the
length of DNA and make the cut at the specific site called
the restriction site. This gives rise to sticky ends in the
sequence. The desired genes and the vectors are cut by the
same restriction enzymes to obtain the complementary
sticky notes, thus making the work of the ligases easy to
bind the desired gene to the vector.

2. Vectors; The vectors help in carrying and integrating

the desired gene. These form a very important part of
the tools of recombinant DNA technology as they are
the ultimate vehicles that carry forward the desired
gene into the host organism. Plasmids and
bacteriophages are the most common vectors
in recombinant DNA technology.

3. Host Organisms: Host organism is the organism

into which the recombinant DNA is introduced. The
host is the ultimate tool of recombinant DNA
technology which takes in the vector engineered with
the desired DNA with the help of the enzymes. There are
a number of ways in which this recombinant DNA’s
are inserted into the host, namely – microinjection,
biolistics or gene gun, alternate cooling and heating,
use of calcium ions, etc.
 Biotechnological
Application in
Medicine
The recombinant DNA technological processes have made
immense impact in the area of healthcare. The most obvious
commercial applications
are diagnosis of disease
and commercial
production of
pharmaceuticals for
disease therapy,
valuable proteins
(enzymes or hormones),
gene therapies and
powerful vaccines. Genetically engineered bacterial cells are
grown in vats to obtain proteins or drugs at commercial
scale such as human insulin, human growth hormone, etc.
RDT enables mass production of safe and more effective
therapeutics do not induce unwanted immunological
responses. At present, about 30 recombinant therapeutics
have been approved globally and 12 of these are presently
being marketed in India
 Recombinant Insulin/Genetically Engineered
Insulin
Insulin, synthesized by the  cells of the islets of
Langerhans in the pancreas, controls the level of glucose in
the blood. An insulin deficiency manifests itself as
diabetes mellitus whose symptoms can be alleviated by a
continuing program of insulin injections, therapy
supplementing the limited amount of hormone synthesized
by the patient’s pancreases. Insulin used for diabetes was
earlier extracted from pancreas of slaughtered cattle and
pigs. Animal insulin is generally satisfactory, though
caused allergy in some patients.

Insulin cannot be orally administered to diabetic

patient because it degrades in alimentary canal.

Two feature that facilitated production of insulin by

recombinant DNA techniques:

 It does not require modification after translation by

the addition of sugar molecules.

 Insulin is a relatively small protein, comprising two

polypeptides, one of 21 amino acid (the A chain) and
the other of 30 amino acids (the B chain) that are
linked by disulphide bonds/bridges.
In Mammals, including humans, insulin is synthesised as
a pro-hormone (like a pro-enzyme, the pro-hormone also
needs to be processed before it becomes a fully mature and
functional hormone) which contain an extra stretch called
 the C peptide. The C peptide is not present in the mature
insulin and is removed during maturation into insulin.
The main challenge for production of insulin using rDNA
techniques was getting insulin assembled into a mature
form. In 1983, Eli Lilly an American Company prepared
two DNA sequences corresponding to A and B, chains of
human insulin and introduced them in plasmids of E.coli
to produce insulin chains. Chains A and B were
produced separately, extracted and combined by
creating disulfide bonds to form human insulin.

 Gene Therapy
If a person is born with a hereditary disease, can a
corrective therapy be taken for such a disease? Gene
therapy is an attempt to do this. Gene therapy is a
collection of methods that allows correction of a gene defect
that has been diagnosed
in a child/embryo. Here
genes are inserted into a
person’s cells and tissues
to treat a disease.
Correction of a genetic
defect involves delivery of
a normal gene into the
individual or embryo to
take over the function of
 and compensate for the non- functional gene.
The gene clinical gene therapy was given in 1990 to a
4year old girl with adenosine deaminase (ADA)
deficiency. This enzyme is crucial for the immune system
to function. The disorder is caused due to the deletion of the
gene for adenosine deaminase. In some children ADA
deficiency can be cured by bone marrow transplantation;
in others it can be treated by enzyme replacement therapy,
in which functional ADA is given to the patient by
injection. But the problem with both of these approaches that
they are not completely curative. As a first step towards
gene therapy, lymphocytes from the blood of the patient are
grown in a culture outside the body. A functional ADA
cDNA (using a retroviral vector) is then introduced into
these lymphocytes, which are subsequently returned to the
patient. However, as these cells are not immortal, the
patient requires periodic infusion of such genetically
engineered lymphocytes. However, if the gene isolate from
marrow cells producing ADA is introduced into cells at
early embryonic stages, it could be permanent cure.

 Molecular Diagnosis:
For effective treatment of a disease, early diagnosis and
understanding the path physiology is very important.
The term diagnosis refers to the act or process of
determining the nature and cause of a disease through
evaluation of patient history, examination and review of
laboratory data. In short, diagnosis is what the disease is
For e.g. you can have a diagnosis of asthma.

Pathophysiology is the study of changes in normal,

mechanical, physical and biochemical functions caused by
a disease
Presence of a pathogen (bacteria viruses) is normally
suspected only when the pathogen has produced disease
symptoms. By this time, the conc. of pathogen is already
very high in the body. Methods of detection fall under two
categories:

Methods of Diagnosis

Traditional Modern

Conventional method Recombinant DNA

technology (RDT)
Known biology or
association of single Polymerase chain
analysts (glucose, Reaction
cholesterol etc) Enzyme linked
Immuno Sorbent
Early detection is not
Assay (Elisa)
possible
Serve purpose of early
Involves serum and urine
diagnosis
analysis
Details of modern methods of diagnosis:
1. PCR:
 It helps to detect very low conc. of bacteria or virus
at the time when the symptoms of the disease are not
visible by amplification of their nucleic acid.
 PCR is now routinely used to detect HIV in
suspected AIDS patient.
 It is being used to detect mutation in gene in
suspected cancer patients too.
 It is powerful technique to identify many other
genetic disorders.

2. ELISA:
 Based on the principle of Antigen-Antibody
interactions.
  Infection by pathogen can be detected by the
presence of antigens (proteins, glycoprotein) or by
detecting the antibodies synthesized against the
pathogen.
 The enzymes frequently used in ELISA include
peroxides and alkaline phosphate.

ELISA is an extremely sensitive test that is used to detect

antibodies (Ab) or specific antigens (Ag). It is carried out
in a well microtiter plate. The direct ELISA is a test for the
presence of antigen. In this procedure, a known antibody is
absorbed to the inside of the well in a microtiter plate. After
rising to remove excess antibody, the sample suspected of
containing the antigen is added. Next, an enzyme linked
Ab that can react with Ag is added. If Ag is put in the well,
 the enzyme linked Ab binds to it and is retained. The
colourless indicates the presence of the Ag.
In indirect ELISA, Ag is added to the microtiter plate well
and the Ag attaches to the walls of the microtiter plate. After
rinsing, to remove excess Ag the serum suspected of
containing the Abs is added. Enzyme linked antibody
capable of reacting with the constant region of other Abs is
then added, followed by addition of the colourless substrate.
Development of colour indicates the presence of Ab being
identified.

 Transgenic Animals:
Animals that had their DNA manipulated to possess and
express an extra gene are known as transgenic animals.
The genome of their animals has been changed and they
can carry genes from other species. Examples of transgenic
animals include rats, rabbits, pigs, sheep, cow, monkey and
fish although over 95% transgenic animals are mice.
How are transgenic animals produced?
1. DNA microinjection
2. Retrovirus-mediated gene transfer
3. Embryonic stem cell mediated gene transfer.
Why are these transgenic animals being produced?
1. Some transgenic animals are produced for specific
economic trait.
2. Other transgenic animals are produced as disease
models (animals genetically manipulated to exhibit
disease symptoms so that effective treatment can be
studied.

Benefits from
transgenic animals
can be studied under 3
heads
Cows that
produce
Medicine human
protein
enriched
milk
Larger
sheep that
Agriculture grow more
wool.
Goats that
produce
Industry spider
milk for
material
production
1. Medicine:
 Normal physiology and development: Transgenic
animals can be specifically designed to allow the
study of how genes are regulated, and how they
affect the normal functions of the body and its
development E.g., Study of complex factors involved
in growth such as insulin-like growth factor. By
introducing genes from other species that alter the
formation of this factor and studying the biological
effects that result, information is obtained about the
biological role of the factor in the body.
 Study of disease: Many transgenic animals are
designed to increase our understanding of how genes
contribute to the development of disease. These are
specially made to serve as models for human diseases
so that investigation of new treatment for diseases is
made possible. Toady transgenic models exist for
many human diseases such as cancer, cystic
fibrosis, rheumatoid arthritis and Alzheimer’s
 Biological Products: Medicines required to treat
certain human diseases can contain biological
products, but such products are often expensive to
make. Transgenic animals that produce useful
biological products can be created by the introduction
of the portion of DNA (or genes) which codes for a
particular product such as human protein (-1-
antitrypsin) used to treat emphysema. Similar
attempts are being made for treatment of
phenylketonuria (PKU) and cystic fibrosis. In 1997,
the first transgenic cow, Rosie, produced human
enriched protein milk. The milk contained the
human alpha- lactalbumin and was nutritionally a
more balanced product for human babies than
natural cow milk
 Vaccine Safety: Transgenic mice are being
developed for use in testing the safety of vaccines
before they are used on
humans. Transgenic
mice are being used to
test the safety of the
polio vaccine. If
successful and found
to be reliable, they
could replace the use of monkeys to test the safety of
batches of the vaccine.
 Chemical Safety Testing: This is known as
toxicity/safety testing. The procedure is the same as
that used for testing toxicity of drugs. Transgenic
animals are made that carry genes which made them
more sensitive to toxic substances than non-
transgenic animals. They are then exposed to the
toxic substances and the effects studied. Toxicity
 testing in such animals will allow us to obtain
results in less time.
2. Agriculture:
I. Breeding: Farmers have
always used selective
breeding to produce animals
that exhibit desired traits.
Traditional breeding is a
time consuming difficult
task. When technology
using molecular biology was developed, it becomes
possible to develop traits in animals in a shorter time
and with more precision. In addition, it offers the
farmer an easy way to increase yields.
II. Quality: Transgenic cows exist that produce more
milk with less lactose or cholesterol, pigs and cattle
that have more meat on them, and sheep that grow
more wool. In the past, farmers used growth hormones
to spur the development of animals but the technique
was problematic, especially since residue of the
hormones remained in the animal product.
III. Disease resistance: Scientists are attempting to
produce disease resistant animals, such as influenza
resistant pigs, but a very limited number of genes
area currently known to be responsible for resistance
to diseases in farm animals.
 Ethical Issues: