CERTIFICATE

This is to certify that Master Rohit Kumar of Class XII-C,

having board roll no.- from school
Kendriya Vidyalaya Hinoo, 1st Shift, Ranchi has
successfully completed the project on “Analysis of the given
samples of Antacids by determining the amount of HCl
they can neutralise” in the academic year 2019-20.

PRINCIPAL SIGNATURE

MR. M.K. SINGH

PGT CHEMISTRY EXTERNAL EXAMINER
K.V.HINOO,1ST SHIFT, RANCHI
 ACKNOWLEDGEMENT

I would like to express my greatest gratitude to the

people who have helped and supported me
throughout this project.

I am grateful to my principal “Miss .S. Toppo” for

her continuous support for the project from initial
advice and contacts in the early stage of
conceptual interception and encouragement to put
these ideas well.

Secondly I would like to thank my biology teacher

“Mrs. Kumkum Shrivastava”{PGT Biology} for her
undivided support and interest who inspired me to
go my own way without whom I would not be able
to complete this project.

I want to thank my parents and friends who

appreciated me and made all things possible.
INDEX
 AIM :
PROCESS OF RECOMINANT DNA
TECHNOLOGY
 INTODUCTION
Biotechnology is a field of applied biology that involves the use of living
things in engineering, technology, medicine, and other useful
applications. Modern use of the term includes genetic engineering as
well as cell and tissue culture technologies.
Recombinant DNA is the technology used for making artificial DNA
(genetic modification) by combining different genetic
materials (DNA) from different sources. It is the laboratory methods of
genetic recombination to get genetic material (DNA) from different
sources which would create a genomic sequence that would not
otherwise be found in the genome.
 1. ISOLATION OF GENETIC MATERIAL
Genetic material of all living organisms is ‘nucleic acid’. In most
organisms, it is DNA, whereas in some it is RNA. The first step in rDNA
technology is to isolate the desired DNA in its pure form i.e. free from
other macromolecules.
However, in a normal cell, the DNA not only exists within the cell
membrane, but is also present along with other macromolecules such as
RNA, polysaccharides, proteins, and lipids. We can use the following
enzymes for specific purposes:
 Lysozyme – to break bacterial cell wall.
 Cellulase – to break plant cell wall.
 Chitinase – to break fungal cell wall.
 Ribonuclease – removes RNA.
 Protease – removes proteins (such as histones that are associated
with DNA).
Other macromolecules are removable with other enzymes or treatments.
Ultimately, the addition of ethanol causes the DNA to precipitate out as
fine threads. This is then spooled out to give purified DNA.
 2. CUTTING OF DNA AT SPECIFIC LOCATIONS
Restriction enzymes act as molecular scissors that cut DNA at specific
locations. These reactions are called ‘restriction enzyme digestions’.
They involve the incubation of the purified DNA with the selected
restriction enzyme, at conditions optimal for that specific enzyme.
The technique – ‘Agarose Gel Electrophoresis’ reveals the progress of
the restriction enzyme digestion. This technique involves running out the
DNA on an Agarose gel. On the application of current, the negatively
charged DNA travels to the positive electrode and is separated out based
on size. This allows us to separate and cut out the digested DNA
fragments. The vector DNA is also processed using the same procedure.
The smaller the fragment size, the farther it moves and the separated DNA
fragments can be visualized only after staining the DNA with a compound
known as Ethidium bromide followed by exposure to UV radiations.
Bright orange colored bands of DNA can be observed in an Ethidium
bromide stained gel exposed to UV light.
The separated bands of DNA are cut out from the Agarose gel and
extracted from the gel piece by the process known as Elution.
3. AMPLIFICATION OF GENE OF INTEREST USING PCR

Polymerase Chain Reaction or PCR is a method of making multiple

copies of a DNA sequence using the enzyme – DNA polymerase. It helps
to amplify a single copy or a few copies of DNA into thousands to millions
of copies. PCR reactions are run on ‘thermal cyclers’ using the following
components:
 Template – DNA to be amplified
 Primers – small, chemically synthesized oligonucleotides that are
complementary to a region of the DNA.
 Enzyme – DNA polymerase
 Nucleotides – needed to extend the primers by the enzyme.

PCR includes three main steps-

1. Denaturation is the process of heating of target DNA at 94C to
separate the two strands of DNA.
2. Annealing is the process of pairing of primers with complimentary
base sequences of the two separated strands.
3. Extension is the process of adding complimentary
deoxyribonucleotides one by one to the 3’OH ends of primers by the
activity of DNA polymerase and as a result new DNA strand is
synthesized.
If the process of replication of DNA is repeated many times, the
segment of DNA can be amplified to approximately billion times by the
use of a thermostable DNA polymerase isolated from a bacterium,
Thermos aquaticus.
The amplified fragment can be used to ligate with a vector for further
cloning.
 4. LIGATION OF DNA MOLECULES
The purified DNA and the vector of interest are cut with the same restriction
enzyme. This gives us the cut fragment of DNA and cut vector, that is now
open. The process of joining these two pieces together using the enzyme
DNA ligase is ligation. The resulting DNA is recombinant DNA.
 5. INSERTION OF RECOMBINANT DNA INTO THE
HOST CELL/ORGANISM
In this step, the recombinant DNA is introduced into a recipient host cell. This
process is ‘Transformation’. Bacterial cells do not accept foreign DNA easily.
Therefore, they are treated to make them ‘competent’ to accept new DNA.
During transformation, if a recombinant DNA bearing a gene for ampicillin
resistance is transferred into recipient E. coli cells, then the E. coli cells also
become ampicillin-resistant. This aspect is useful in differentiating
transformed cells from non-transformed cells.
For example, if we spread the transformed cells on agar plates containing
ampicillin, only the transformed, ampicillin-resistant cells will grow while the
untransformed cells will die. Therefore, in this case, the ampicillin resistance
gene acts as the ‘selectable marker’.

6. OBTAINING THE FOREIGN GENE PRODUCT

The recombinant DNA multiplies in the host and is expressed as a protein,
under optimal conditions. This is now a recombinant protein.
Small volumes of cell cultures will not yield a large amount of
recombinant protein. Therefore, large-scale production is necessary to
generate products that benefit humans. For this purpose, vessels
called bioreactors are used.
Bioreactors are large containers with a continuous culture system, where
the fresh medium is added from one side and used medium is taken out
from another side.
Bioreactors can process about 100-1000 litres of cell cultures.
A bioreactor provides optimum conditions (temperature, oxygen, pH,
vitamins etc.) to biologically convert raw materials into specific proteins,
enzymes etc.
‘Stirred-tank bioreactor’ is the most common type of bioreactor. It is
usually cylindrical and has the following parts:
 Agitator system – to stir the contents evenly.
 Oxygen delivery system – to introduce air into the system.
 Foam control system
 Temperature control system
 pH control system
 Sampling ports – to take out small amounts of culture.

7. DOWNSTREAM PROCESSING
Before the protein is marketed as a final product, it is subjected to
downstream processing which includes:

 Separation and purification.

 Formulation with suitable preservatives.
 Clinical trials to test the efficacy and safety of the product.
 Quality control tests.
 CONCLUSION

Recombinant DNA technology is an important development in

science that has made the human life much easier. In recent years, it has
advanced strategies for biomedical applications such as cancer
treatment, genetic diseases, diabetes, and several plant disorder
especially viral and fungal resistances. The recombinant DNA
technology enhanced resistance of plants to different adverse acting
factors has been recognized widely. The improvement it brought not only
for humans but also in plants and microorganisms are very significant.
The recombinant technology is helping in treating several diseases
which cannot be treated in normal conditions, although the immune
responses hinder achieving good results.
Several difficulties are encountered by the genetic engineering
strategies which needed to be overcome by more specific gene
enhancement according to the other organism’s genome. The
introduction of genetic material from one source into the other is a
disaster for safety and biodiversity. There are several concerns over
development of genetically engineered plants and other products.
Further, concerns exist that genetic engineering has dangerous health
implications. Thus, further extensive research is required in this field to
overcome such issues and resolve the concerns of common people.
 BIBLIOGRAPHY
Biology Text Book Class XII, NCERT

WEBSITES-
www.google.com
en.wikipedia.org