RECOMBINANT DNA TECHNOLOGY techniques and steps and applications

1. Recombinant DNA
technology
APPLICATIONS- rDNA TECHNOLOGY
Presentation By:
AZMATHULLAH.R.
M.Sc.MGMD – Ist Yr
Reg No : 31221701
Sub - Recombinant DNA Technology

2. OVERVIEW:-
 WHAT IS DNA?
 STRUCTURE OF DNA
 FUNCTIONS OF DNA
 DEFINITION Recombinant DNA
 How is Recombinant DNA made? History
 How does rDNA work?
 Why is rDNA important
 APPLICATIONS
 LIST OF NEWER DRUGS
 ADVANTAGES & DISADVANTAGES
 BIBLOGRAPHY

3. WHAT IS DNA?
 Deoxyribonucleic acid (abbreviated DNA) is the
molecule that carries genetic information for the
development and functioning of an organism.
 DNA is made of two linked strands that wind around
each other to resemble a twisted ladder — a shape
known as a double helix.
 Each strand has a backbone made of alternating sugar
(deoxyribose) and phosphate groups.

4.  Attached to each sugar is one of four bases: adenine
(A), cytosine (C), guanine (G) or thymine (T).
 The two strands are connected by chemical bonds
between the bases: adenine bonds with thymine, and
cytosine bonds with guanine. (A=T ; C=G)
 The sequence of the bases along DNA’s backbone
encodes biological information, such as the
instructions for making a protein or RNA molecule.

5. STRUCTURE - DNA

7. The discovery in 1953 of the double helix, the
twisted-ladder structure of deoxyribonucleic acid (DNA),
by James Watson and Francis Crick.
DISCOVERY :-

8. FUNCTIONS – DNA
 DNA is the information molecule. It stores instructions for
making other large molecules, called proteins.
 These instructions are stored inside each of your cells, distributed
among 46 long structures called chromosomes.
 These chromosomes are made up of thousands of shorter
segments of DNA, called genes.
 Replication.
 Mutation & Recombination
 Gene Expression

9. DEFINITION– rDNA
 Genetics recombination is the exchange
information between two DNA segments.
 This is common occurence in the same species
 But by artificial means, when a gene of one
species is transferred to another living organism, it
is called as recombinant DNA
technology(rDNA).

10.  Production of a unique DNA molecule together two or
more DNA fragments not normally associated with
each other
 DNA fragments are usually derived from different
biological sources
 A recombinant DNA molecule can enter a cell and
replicate.
 Recombinant DNA, also known as Chimeric DNA.

11.  Contains sequences taken from very different sources, Eg.
DNA containing both human & bacterial DNA sequences
The development of these new technologies have resulted
into production of large amount of biochemically products.
• r-DNA involves using microorganisms:-
1. To create new pharmaceuticals
2. To create safer/ more effective version therapeutic agents
Three different methods of DNA recombination..
i. Transformation
ii. Non-bacterial transformation
iii. Phage induction

13. HISTORY – rDNA
 Recombinant DNA technology is one of the recent
advances in science, which was developed by two
scientists named Boyer & Cohen in 1973.
 This is common occurence in the same species

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

16. ROLE OF ENZYMES –rDNA
TECHNOLOGY
♦ RESTRICTION ENDONUCLEASE:
Cleaves DNA 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 3' ends (removes RNA
polymer).

17. ♦ 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.

18. BASIC – DEFINITIONS
CLONING:-
 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.

19. PLASMIDS:-
These are double stranded DNA that are usually circular and
mostly found inside certain bacterial species. 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.

20. STEPS - rDNA TECHNOLOGY
Isolation of DNA
CuttingDNAatpreciselocations
Joiningtwo DNA fragmentscovalently
Selection ofasmallmolecule ofDNA capableof self-replication
Movingrecombinantmolecules fromthetest tubeinto ahost cell
Selecting oridentifyingthose cellsthatcontainRecombinantDNA

21. 1) Isolation of DNA
• The first step involves isolating
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.

22. 2) Cleavage of DNA
• Special enzymes, Restriction enzymes cut
DNA chains at specific locations.
• These enzymes are also called "Molecular
scissors“.
• The specific site is called "Restriction site"
which is 4-7 base pair long.
• The fragments of DNA obtained after the
action of restriction enzymes are called
"Restriction fragments"; they can have
sticky or blunt ends.

23. 3) Joining two DNA fragments covalently
• The DNA fragments are joined together by DNA
ligase.

24. 4) Moving recombinant molecules
from the test tube into a host cell
E. coli is the most common host cell due to
the following advantages:
• It's DNA metabolism is well understood
• Cloning vectors associated with E.Coli
are well characterized
• Effective techniques are available for
moving DNA from one bacterial cell to
another

25. 5) Selecting or identifying
those cells that contain
Recombinant DNA
 Colony or plaque hybridization is
the method by which specific
clones are identified and purified.
 Bacteria are grown as colonies on
an agar plate.

26. APPLICATIONS - rDNA TECHNOLOGY
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.

27. 8. Many monoclonal antibodies
9. Pharmaceutical & therapeutic applications
10. Gene therapy
11. Medical diagnosis
12. Xenotransplants, etc.,

28. HUMULIN (INSULIN)
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
pancrease islet’s of Langerhans, regulates the use & 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.

30. HEPATITIS B VACCINE (HB)
HEPATITIS B is an infectious inflammatory illness of the
liver caused by hepatitis B virus (HBV)
HEPATITIS B VACCINE:
It is sterile solution of Immunoglobulin containing
antibody to hepatitis B surface antigen.
Kept @ pH 6.2
Formulated in 0.075M NaCl, 0.15M glycine, 0.01%
polysorbate 80
Used by i.m.route as a vaccine for hepatitis B.

31. PREPARATION OF HEPATITIS B
VACCINE BY r-DNA
Hepatitis B vaccine (rDNA) is produced by the
expression of the viral gene coding foe HBs Ag in yeast
(Saccharomyces cerevisiae)
Or
Mammalian cells (Chinese Hamster Ovary (CHO) cells or
other suitable cell lines).

32. 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 /NaOH
added to adjust pH upto 7.5.

33. LIST OF SOME
OTHER NEWER
DRUGS - PREPARED
BY r-DNA
TECHNOLOGY

34. 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.

35. 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.

36. HepatitisB surface antigen Vaccination (now in market)
Interleukin-2 Treatment of cancer
Tissue Plasminogen Activator Thrombosis
Erythropoetin Treatment of Anemia

37. Some of the areas where
Recombinant DNA are
used.

38. Recombinant DNA has been gaining in importance over the last few
years, & rDNA will only become more important in the 21st century
as genetic diseases become more prevalent & 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 & cure of sickle cell anemia
• Prevention & 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

39. Advantages Disadvantages
Provide substantial quantity Prone to cause Mutation
No need for natural or organic
factors
Effect natural immune system
Unlimited utilizations Can destroy natural ecosystem
Cheap Non economical for every
people
Resistant to natural inhibitors Commercialized & became
big source of income

40. BIBLOGRAPHY
• Pharmaceutical Biotechnology - Fundamentals And Applications
• Biochemistry-U. Satyanarayana
• A Text Book of Biotechnology-R .C Dubey
https://en.wikipedia.org/wiki/PBR322
https://www.slideshare.net/TapeshwarYadav1/recombinant-dna-technology-49722102
https://www.slideshare.net/namarta28/recombinant-dna-technology-part-1pdf
https://www.slideshare.net/seetaram443/recombinant-dna-rdna-technology
https://www.slideshare.net/bharathpharmacist/recombinant-dna-technology-1
https://www.slideshare.net/TapeshwarYadav1/recombinant-dna-technology-49722102

41. "What the public needs to understand is that these new technologies, especially in
recombinant DNA technology, allow scientists to bypass biological boundaries