This document summarizes a student's submission on the topic of recombinant DNA technology. It discusses various topics related to rDNA technology including DNA, genes, plasmids, how recombinant DNA is made using transformation and other methods, use of restriction enzymes and ligases, gene therapy applications, and recent advances in using gene therapy to treat brain cancer and developing breast cancer treatments. The submission provides an overview of the key concepts and techniques in recombinant DNA technology.

1. DEPARTMENT OF PHARMACEUTICAL SCIENCES,
KUMAUN UNIVERSITY
SUBMITTED BY:
SHWETA SINGH
M.PHARM 1ST YEAR
1
SUBMITTED TO:
Dr. Anita Singh
Senior Asst. Professor
TOPIC – RECOMBINANT DNA
TECHNOLOGY

2. Topics for Discussion
oBrief study of rDNA Technology
oHybridoma Technology
oGene Therapy
oClinical Application & Recent Advances in Gene Therapy
2

3. DNA
 DNA is the keeper of the all information
needed to recreate an organism
Nucleotides are the building blocks of the
DNA
All DNA is made up of a base consisting
of sugar phosphates and nitrogen bases.
“Double helix"
 The sugar used in DNA is deoxyribose.
DNA contain a anti-parallel strands.
DNA contains 4 nitrogen bases they are:
 Purines: Adenine, Guanine
Pyrimidines: Thymine, Cytosine
They are found in pairs, A&T and G&C
3

4. GENE
A gene is a stretch of DNA that codes for a type of protein that has a
function in the organism.
 It is a unit of heredity in a living organism. All living things depend on
genes.
Genes hold the information to build and maintain an organism’s cell
and pass genetics traits to off spring.
 DNA does not actually make the organism, it only makes proteins.
The DNA is transcribed into mRNA and mRNA is translated into
protein, and the protein then forms the organism. (Central Dogma)
4

5. PLASMID
 These are double stranded DNA that are usually circular and mostly
found inside certain bacterial specie e.g. E.coli.
However most plasmids are now commercially available, ready to be
used, providing specific fragment insertion sites.
 Plasmids in genetic engineering are also known as ‘vectors’.
Vectors also include viruses known as bacterio‐phage that use
bacteria as their host to replicate.
 Hence a bacterio‐phage can be used to transfect and create several
copies of the DNA fragment of interest by replicating several times in a
bacteria.
5

6. Recombinant DNA Technology
(rDNA)
 Molecules of DNA from two different species that are inserted into a
host organism to produce new genetic combinations that are of value
to science, medicine, agriculture, and industry.
For example : DNA comprising an animal gene may be recombined
with DNA from a bacterium.
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7. Recombinant DNA (rDNA)
7
DNA molecules constructed outside of living cells by
joining natural or synthetic DNA segments to DNA
molecules that can replicate in a living cell.
The DNA is inserted into another DNA molecule
called ‘vector’ .The recombinant vector is then
introduced into a host cell where it replicates itself,
the gene is then produced.

8. Basic Principles of rDNA Technology
Generation of DNA fragments & selection of the desired
piece of DNA.
Insertion of the selected DNA into a cloning vector to create
a rDNA or chimeric DNA.
Introduction of the recombinant vectors into host cells.
Multiplication & selection of clones containing the
recombinant molecules.
Expression of the gene to produce the desired product. 8

9. 9

10. How is
Recombinant
DNA made?
There are three different methods
by which Recombinant DNA is
made:
Transformation
Phage Introduction
 Non-Bacterial Transformation. 10

11. Transformation
 Transformation is the process by which exogenous DNA is
transferred into the host cell. Transformation usually implies uptake
of DNA into bacterial, yeast or plant cells, while transfection is a term
usually reserved for mammalian cells.
The first step in transformation is to select a piece of DNA to be
inserted into a vector.
The second step is to cut that piece of DNA with a restriction
enzyme and then ligate the DNA insert into the vector with DNA
Ligase. The insert contains a selectable marker which allows for
identification of recombinant molecules. The vector is inserted into a
host cell, in a process called transformation.
One example of a possible host cell is E. coli. The host cells must be
specially prepared to take up the foreign DNA.
11

12. Phage Introduction
Phage introduction is the process of transfection, which is
also known as transformation, except a phage is used
instead of bacteria.
 In vitro packaging of a vector is used. This uses lambda or
MI3 phages to produce phage plaques which contain
recombinants.
The recombinants that are created can be identified by
differences in the recombinants and non-recombinants using
various selection methods.
12

13. Non-Bacterial transformation
Microinjection, the DNA is injected directly into the
nucleus of the cell being transformed.
The host cells are bombarded with high velocity
micro-projectiles, such as particles of gold or
tungsten that have been coated with DNA.
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14. Restriction Endonucleases
Enzymes for the manipulation of DNA.
Are bacterial enzymes that can cut/split DNA at specific
sites.
These were first discovered in E.coli restricting the
replication of bacteriophages,by cutting the viral DNA(The
host E.coli DNA is protected from cleavage by addition of
methyl groups).Thus,the enzymes that restrict the viral
replication are known as restriction enzymes or restriction
endonucleases.
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15. 15

16. DNA ligases
These were originally isolated from viruses, also occur in
E.coli & eukaryotic cells.
 The cut DNA fragments are covalently joined together by
DNA ligases.
DNA ligase joins the DNA fragments by forming a
phosphodiester bond b/n the phosphate group of 5’-carbon
of one deoxyribose with the hydroxyl group of 3’-carbon of
another deoxyribose.
16

17. Steps involved in rDNA
Technology
17

18. Steps involved in rDNA
Technology
1. Isolation of Genetic Material
The first step in rDNA technology is to isolate the desired
DNA in its pure form i.e. free from other macromolecules.
Since DNA exists within the cell membrane along with
other macromolecules such as RNA, polysaccharides,
proteins, and lipids, it must be separated and purified which
involves enzymes such as lysozymes, cellulase, chitinase,
ribonuclease, proteases etc.
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
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19. 2. Restriction Enzyme Digestion
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 separating and cutting out the
digested DNA fragments.
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20. 3. Amplification Using PCR
 Polymerase Chain Reaction or PCR is a method of
making multiple copies of a DNA sequence using the
enzyme – DNA polymerase in vitro.
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:
i. Template – DNA to be amplified
ii. Primers – small, chemically synthesized oligonucleotides
that are complementary to a region of the DNA.
iii. Enzyme – DNA polymerase
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21. iv. Nucleotides – needed to extend the primers by the
enzyme.
The cut fragments of DNA can be amplified using PCR
and then ligated with the cut vector.
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22. 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 the cut vector,
that is now open.
The process of joining these two pieces together using the
enzyme ‘DNA ligase’ is ‘ligation’.
The resulting DNA molecule is a hybrid of two DNA
molecules – the interest molecule and the vector. In the ter-
minology of genetics this intermixing of different DNA strands
is called recombination.
22

23. Hence, this new hybrid DNA molecule is also called a
recombinant DNA molecule and the technology is referred to
as the recombinant DNA technology.
23

24. 5. Insertion of Recombinant DNA Into Host
In this step, the recombinant DNA is introduced into a
recipient host cell mostly, a bacterial 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. The processes used may be thermal shock, Ca++ ion
treatment, electroporation etc.
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25. 6. Isolation of Recombinant Cells
The transformation process generates a mixed population
of transformed and non-trans- formed host cells.
The selection process involves filtering the transformed
host cells only.
For isolation of recombinant cell from non-recombinant
cell, marker gene of plasmid vector is employed.
For examples, PBR322 plasmid vector contains different
marker gene (Ampicillin resistant gene and Tetracycline
resistant gene. When pst1 RE is used it knock out Ampicillin
resistant gene from the plasmid, so that the recombinant cell
become sensitive to Ampicillin.
25

26. Application of Recombinant DNA
technology
1. The most common application of recombinant DNA is in
basic research, in which the technology is important to
most current work in the biological and biomedical
sciences.
2. Recombinant DNA is used to identify, map and sequence
genes, and to determine their function.
3. Recombinant proteins are widely used as reagents in
laboratory experiments and to generate antibody probes
for examining protein synthesis within cells and
organisms.
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27. 4. Many additional practical applications of recombinant
DNA are found in industry, food production, human and
veterinary medicine, agriculture, and bioengineering.
5. DNA technology is also used to detect the presence of
HIV in a person.
6. Application of recombinant DNA technology in
Agriculture – For example, manufacture of Bt-Cotton to
protect the plant against ball worms.
7. Application of medicines – Insulin production by DNA
recombinant technology is a classic example.
8. Gene Therapy – It is used as an attempt to correct the
gene defects which give rise to heredity diseases.
9. Clinical diagnosis – ELISA is an example where the
application of recombinant DNA is possible.
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28. Hybridoma Technology
Hybridoma technology is a method for producing large
numbers of identical antibodies (also called monoclonal
antibodies).
This process starts by injecting a mouse (or other
mammal) with an antigen that provokes an immune
response. A type of white blood cell, the B cell, produces
antibodies that bind to the injected antigen.
These antibody producing B-cells are then harvested from
the mouse and, in turn, fusedwith immortal B cell cancer
cells, a myeloma to produce a hybrid cell line called
a hybridoma, which has both the antibody-producing ability
of the B-cell and the longevity and reproductivity of the
myeloma.
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29. 29

30. MONOCLONAL ANTIBODY
 Monoclonal antibodies (mAb)
are antibodies that are identical
because they are produced by
one type of immune cell, all
clones of a single parent cell.
 Basically produced by white
blood cell which is called as
plasma cell.
 Is used for treatment of
cancerous cells and as anti-
venom ( anti snake venom)
30

31. PROCEDURE :
1. Immunization of specific animal which generate
hybridoma cell with spleen cell.
2. Isolation of myeloma cells.
3. Fusion between spleen cell and myeloma cell.
4. Selection of HAT medium.
5. Isolation of hybridoma cell.
6. Screening of hybridoma cell
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32. 1. Immunization of specific animal
An antigen immunized to an animal (like mice) via
intravenously(directly to blood) by injection.
Where in spleen it activate B-cell which produce plasma cell
(spleen cell).
Plasma cell to produce monoclonal antibodies
Isolation of plasma cell from spleen of animal pecific
animal
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33. 2. Isolation of myeloma cells.
 Myeloma cells are cancerous cells which is isolated from
bone-marrow.
Myeloma cells are generally immortal in nature (that which
never dies) and has multiplication property.
3. Fusion of spleen cell and myeloma cell
It requires PEG (poly ethylene glycone) medium for fusion
 It can also done by electro fusion.
Fusion between spleen cell and myeloma cell produced
five different types of cells.
1. Fused plasma 4. Fused myeloma
2. Hybridoma 5. Unfused plasma
3. Unfused myeloma
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34. Application of Hybridoma Technology
Serological
Identification of ABO blood group
Diagnosis
• Detection of pregnancy by assaying of hormones with
monoclonal.
•Separation of one substance from a mixture of very similar
molecules.
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35. Immunopurification
•Purification of individual interferon using monoclonal.
•Inactivation of T-lymphocytes responsible for rejection of
organ transplants.
Therapy
• Removal of tumor cell from bone marrow.
•Treatment of acute renal failure.
•Treatment malignant leukemic cells, B cell lymphomas, and
a variety of allograft rejections after transplantation.
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36. Gene Therapy
 Gene therapy is the insertion of genes into an individual's
cells and tissues to treat a disease, such as a hereditary
disease in which a deleterious mutant allele is replaced with
a functional one.
 Although the technology is still in its infancy, it has been
used with some success.
Researchers are studying gene therapy for a number of
diseases, such as
1. Severe combined immuno-deficiencies (SCID)
2. Hemophilia 4. HIV
3. Parkinson's disease 5. Cancer
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37. How it work ?
1. A vector delivers the therapeutic gene into a patient‟s
target cell
2. The target cells become infected with the viral vector
3. The vector‟s genetic material is inserted into the target
cell
4. Functional proteins are created from the therapeutic
gene causing the cell to return to a normal state
37

38. 38

39. 39

40. 40

41. Clinical Application & Recent
Advances in Gene Therapy
First Real-Time MRI-Guided Gene Therapy for Brain
Cancer
Neurosurgeons at the University of California, San Diego
School of Medicine and UC San Diego Moores Cancer
Center are among the first in the world to utilize real-time
magnetic resonance imaging (MRI) guidance for delivery of
gene therapy as a potential treatment for brain tumors.
Using MRI navigational technology, neurosurgeons can
inject Toca 511 (vocimagene amiretrorepvec), a novel
investigational gene therapy, directly into a brain malignancy
41

42. The new approach offers a precise way to deliver a
therapeutic virus designed to make the tumor susceptible to
cancer-killing drugs
Toca 511 is a retrovirus engineered to selectively replicate
in cancer cells, such as glioblastomas.
Toca 511 produces an enzyme that converts an anti-fungal
drug, flucytosine (5-FC), into the anti-cancer drug 5-
fluorouracil (5-FU).
After the injection of Toca 511, the patients are treated with
an investigational extended- release oral formulation of 5-FC
called Toca FC.
Cancer cell killing takes place when 5-FC comes into
contact with cells infected with Toca 511.
42

43. UCLA researchers combine cellular and gene
therapies to develop treatment for breast cancer
Carol Kruse, a professor of neurosurgery and member of
the Jonson Cancer Center and the UCLA Brain Research
Institute led the research on breast cancer
Breast cancer is the most common form of cancer in
women, and metastasis is a major cause of health
deterioration and death from the disease
Cellular therapy and gene therapy were used together to
treat breast cancer
Cellular therapy is a type of immunotherapy that uses T
cells, the foot soldiers of the immune system, that have been
sensitized in the laboratory to kill breast cancer cells.
43

44. These sensitized T cells are injected into the parts of the
brain to which cancer has spread.
The research shows that the T cells can move through
tissue and recognize and directly kill the tumor cells
44

45. Mucopolysaccharidosis Type IIIA potential gene
therapy
Mucopolysaccharidosis Type IIIA (MPSIIIA) is a metabolic
disorder in which the body is missing an enzyme that is
required to break down long chains of sugars known as
glycosaminoglycans .
The glycosaminoglycans collect in the body and cause
damage, particularly in the brain if not broken.
Fàtima Bosch and colleagues at Universit at Autònoma de
Barcelona in Spain developed a form of gene therapy to
replace the enzyme that is missing in MPSIIIA
They injected the replacement gene into the cerebrospinal
fluid that surrounds the brain and spinal cord .
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46. This study demonstrates that gene therapy can be delivered
to the brain through the cerebrospinal fluid and suggests that
this approach could potentially be used as a therapy for
MPSIIIA.
46

47. Referances
1. Verma, P. S., & Agrawal, V. K. (2006). Cell Biology, Genetics, Molecular Biology, Evolution & Ecology (1 ed.). S .Chand and
company Ltd.
2. Klug, W. S., & Cummings, M. R. (2003). Concepts of genetics. Upper Saddle River, N.J: Prentice Hall.
3. https://byjus.com/biology/recombinant-dna-technology/
4. https://en.wikipedia.org/wiki/Recombinant_DNA
5. https://www.britannica.com/science/recombinant-DNA-technology
6. https://www.quora.com/What-are-the-advantages-and-disadvantages-of-recombinant-DNA
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48. Thank you