Recombinant DNA (rDNA) technology involves combining DNA molecules from different sources into a single recombinant DNA molecule. This is done using restriction enzymes to cut the DNA at specific sites and DNA ligase to join the fragments. The resulting recombinant DNA can be inserted into a host cell that will replicate it, allowing mass production of useful proteins like insulin, growth hormone, and monoclonal antibodies for therapeutic use. While rDNA technology has generated many medical advances, it also raises safety and ethical concerns that must be carefully considered and addressed.

1. RECOMBINANT
DNA (r-DNA)
TECHNOLOGY
(Therapeutic Agents Developed From
Recombinant DNA Technology)
S.SEETARAM SWAMY, M.Pharm.,
Asst. professor,
Dept. of Pharmaceutical Chemistry,
Chilkur Balaji College of Pharmacy.
E-mail:seetaram.443@gmail.com

2.  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
DNA – THE GENETIC SECRET
DNA is the keeper of the all the information
needed to recreate an organism.

3.  Prokaryotic (bacteria) or eukaryotic (yeast, mammalian cell culture) systems are
generally used as a host for the production of usable quantities of the desired r-DNA
product.
 Most of the r-DNA products approved by FDA are being produced using these
systems.
Bacteria such as Escherichia coli are
widely used for the expression of r-DNA
products.
 They offer several advantages due to
high level of recombinant protein
expression, rapid growth of cell and simple
media requirement.
Yeast such as Saccharomyces cerevisiae,
Hansenulla polymorpha and Pichia
pastoris are among the simplest eukaryotic
organisms.
 They grow relatively quickly and are
highly adaptable to large-scale production.
 These organisms do not produce
endotoxin.
Mammalian systems such as Chinese hamster ovary (CHO) cell and Baby hamster
kidney (BHK) cell systems are often the choice for production of human therapeutic proteins.

4. 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.
GENE

5. 1970: Hamilton Smith, at Johns Hopkins
Medical School, isolates the first
restriction enzyme, an enzyme that cuts DNA
at a very specific nucleotide sequence.
Herbert Boyer
Stanley Cohen
1978: Somatostatin, which regulates human
growth hormones, is the first human protein
made using recombinant technology.
1972: Stanley Cohen and Herbert Boyer
combine their efforts to create recombinant
DNA.
History of Recombinant DNA Technology

6. DNA that has been created artificially (not natural). DNA from two or
more sources is incorporated into a single recombinant molecule
Recombinant DNA(rDNA) is a form of artificial DNA that is created by
combining two or more sequences. It is made possible by two important
enzymes. Restriction enzymes and DNA Ligase are the two principal tools,
first used by Paul Berg in 1972,employed to alter DNA.
 METHODS BY WHICH RECOMBINANT DNA IS MADE ARE:
TRANSFORMATION
TRANSDUCTION
CONJUGATION
RECOMBINANT DNA (r DNA)

7. Recombinant DNA technology
r-DNA involves using microorganisms
1. To create new pharmaceuticals
2. To create safer/ more effective version therapeutic agents
Recombinant DNA (r-DNA) technology has made a revolutionary impact in the
area of human healthcare by enabling mass production of safe, pure and effective
r-DNA expression products.
•Recombinant DNA is specifically from two or more DNA incorporated into a
single molecule.
• Genetic engineering, recombinant DNA technology, genetic modification and
gene splicing are terms are applied to the direct manipulation of an organisms
gene.
• The development of these new technologies have resulted into production of
large amount of biochemically products

8. Production of a unique DNA molecule by joining together or
more DNA fragments not normally associated with each other,
which can replicate in the living cell.
Recombinant DNA is also called chimeric DNA.
3 different methods of DNA recombination…
A. Transformation
B. Non-bacterial transformation
C. Phage induction

9. RESTRICTION ENDONUCLEASE:
Cleaves DNA at a specific base sequence.
DNA LIGASE:
Binds two DNA molecules or fragments.
DNA POLYMERASE:
Fills single – stranded gaps in duplex DNA by stepwise addition of nucleotides
to 3’ ends (removes RNA polymer).
REVERSE TRANSCRIPTASE:
Makes a DNA polymer of an RNA polymer
POLYNUCLEOTIDE KINASE:
Adds a phosphate to the 5’ OH end of a polynucleotide, to label it or permit
ligation
ALKALINE PHOSPHATASE:
Removes terminal phosphates from the 5’end, the 3’ end, or both.
ROLE OF ENZYMES IN r-DNA TECHNOLOGY

10. © The term ‘clone’ means, exact copy of the parent. A duplicate or a look alike
carrying the same genetic signature or genetic map.
© Cloning is the best application of recombinant DNA technology and could be
applied to something as simple as DNA fragment or a larger, sophisticated
mammalian specie such as humans.
Molecular cloning is carried out in‐vitro where a specific fragment of DNA is
isolated from an organism ‘donor’ and introduced into a ‘plasmid’ that replicates in
a ‘host’ cell making multiple copies of that DNA fragment.
CLONING

11.  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.
PLASMIDS

12. 1. This "sticky ends" from two
different DNA molecules can
hybridize together; then the
nicks are sealed using ligase.
2. The result is recombinant
DNA.
3. When this recombinant vector
is inserted into E. coli, the cell
processes the instructions and
by translation & transcription,
it assembles the amino acids
forming the protein product of
interest.
4. More importantly, the new
instructions are passed along to
the next generation of E. coli
cells forming ‘recombinant
clones’ on the culture agar.

13. Recombinant r-DNA technology
Isolation of the gene of interest
Preparation of vector DNA and DNA to be cloned
Insertion of the gene to the vector molecule and ligation
Introduction of the vector DNA to the appropriate host cell
Amplification of the recombinant DNA molecule in the host cell

14. Recombinant DNA
technology for production
of human therapeutics.

15. Provide substantial quantity
No need for natural or organic factors
Unlimited utilizations
Cheap
Resistant to natural inhibitors
Advantages of Recombinant Technology

16. Commercialized and became big source of income for businessman.
Effects natural immune system of the body.
Can destroy natural ecosystem that relies on organic cycle.
Prone to cause mutation that could have harmful effects.
Major international concern : manufacturing of biological weapons
such as botulism & anthrax to target humans with specific genotype.
Concerns of creating super – human race
Disadvantages of recombinant technology

17. 1. Insulin for diabetics.
2. Factor VIII for males suffering from haemophilia A.
3. Factor IX for haemophilia B.
4. Human growth hormone (HGH).
5. Erythropoietin (EPO) for treating anemia.
6. Several types of interferon.
7. Granulocyte –macrophage colony-stimulating factor(GSM-CSF) for
stimulating the bone marrow after a bone marrow transplant.
8. Many monoclonal antibodies
9. Pharmaceutical and therapeutic applications
10. Gene therapy
11. Medical diagnosis
12. Xenotransplants
Applications of recombinant DNA technology

18.  Since Banting and Best discovered the hormone,
insulin in 1921 diabetic patients, whose elevated sugar
levels are due to impaired insulin production.
 The hormone, produced and secreted by the beta cells
of the pancreas' islets of Langerhans, regulates the use
and storage of food, particularly carbohydrates.
 Chemically, insulin is a small, simple protein.
It consists of 51 amino acid, 30 of which constitute one
polypeptide chain, and 21 of which comprise a second
chain.
 The two chains are linked by a disulfide bond.
HUMULIN (INSULIN)

20.  Hepatitis B vaccine (rDNA) is produced by the expression of the viral
gene coding for HBsAg in yeast (Saccharomyces cerevisiae)
or
mammalian cells (Chinese hamster ovary (CHO) cells or other suitable
cell lines).
HEPATITIS B VACCINE - (HB)
PREPARATION OF HEPATITIS B VACCINE BY r-DNA
HEPATITIS B is an infectious inflammatory
illness of the liver caused by the hepatitis B virus
(HBV).
HEPATITIS B VACCINE:
 It is a sterile solution of Immunoglobin
containing antibody to hepatitis-B surface antigen.
 Kept at p H 6.2
 Formulated in 0.075M NaCl, 0.15M glycine,
0.01% polysorbate 80.
 Used by intramuscular route as a vaccine for
hepatitis B.

21. HUMAN GROWTH HORMONE(SOMATOTROPIN/ HUMATOTROPIN)
 Polypeptide hormone of rdna origin,with 191 amino acids, molecular
weight-2-115 dalton.It is a pituitary hormone.
 Humatroph,a sterilised lauphilised powder sub-cutaneous or i.v. injection.
 Phosphoric acid or NaOH added to adjust pH upto 7.5 and oxygen sensitive.
 Recombinant human growth hormone is generally produced by inserting the human
growth hormone gene into plasmids of E.coli bacteria.
 Recombinant bacteria cells are cultured and human growth hormones produced by these
bacteria are extracted from the extracellular media.
 During the production of the human growth hormone in the body it is attached with
signal peptides with 26 amino acids. This signal peptide is cleaved in the body before
releasing the hGH to do their biological function
 During the production of recombinant human growth hormone the signal peptides
interrupts their production.First cDNA is produced using m RNA encoding for hGH. Then
cDNA are integrated into the plasmid and are inserted into the E.coli bacteria,then they are
cultured and HGH are extracted along with the signal peptide chains.
PREPARATION OF HGH by rDNA

23. LIST OF SOME OTHER NEWER DRUGS PREPARED BY R-DNA
TECHNOLOGY
DRUGS APPLICATIONS
Epogen/Procrit For patients with anemia due to Dialysis/ Chronic Kidney Disease /
Renal Failure / Chemo / HIV
Neulasta For Neutropenia: low WBC count febrile neutropenia (low WBC count
with fever/ infection) due to chemo, BMT, AML
Infergen For patients with Chronic, non-responding, or relapsing hepatitis C viral
(HCV) infection.
Avonex Treatment of relapsing forms of MS. Slows the progression of MS by
regulating the body's immune response against myelin.
Betaseron Multiple Sclerosis: Significantly delays the progression of secondary
MS, including relapsing-remitting MS
Forteo Treatment of osteoporosis in women and men
IntronA treat different types of leukemia,malignant melanoma, multiple
myeloma,basal cell carcinoma.

24. DRUGS APPLICATIONS
HepatitisB surface antigen Vaccination (now in market)
Interleukin-2 Treatment of cancer
Tissue Plasminogen
Activator
Thrombosis
Erythropoetin Treatment of Anemia
LIST OF SOME OTHER NEWER DRUGS PREPARED BY
R-DNA TECHNOLOGY

26. Some recombinant proteins that are used therapeutically

27. Applications of recombinant DNA technology

28. Recombinant DNA has been gaining in importance over the last few years, and
recombinant DNA will only become more important in the 21st century as genetic
diseases become more prevalent and agricultural area is reduced. Below are
some of the areas where Recombinant DNA will have an impact.
 Better Crops (drought & heat resistance)
Recombinant Vaccines (ie. Hepatitis B)
 Prevention and cure of sickle cell anemia
 Prevention and cure of cystic fibrosis
 Production of clotting factors
 Production of insulin
 Production of recombinant pharmaceuticals
 Plants that produce their own insecticides
Germ line and somatic gene therapy

29. REFERENCES
• Pharmaceutical Biotechnology – Fundamentals And Applications
• Biochemistry –U. Satyanarayana
• A Text Book of Biotechnology – R .C Dubey
• A Text book of intermediate second year BOTANY
• https://en.wikipedia.org/wiki/PBR322

30. “What the public needs to understand is that these new technologies, especially
in recombinant DNA technology, allow scientists to bypass biological boundaries
altogether.” ―Jeremy Rifkin