Recombinant DNA technology involves manipulating DNA from different sources to produce novel DNA molecules. It has several key steps: isolating the desired DNA and vector, joining them using enzymes to create recombinant DNA, introducing this into a host cell, and selecting cells that express the gene. This technology has many applications including producing human insulin and growth hormones through bacteria, developing vaccines by cloning genes for antigens, and creating monoclonal antibodies. It allows mass production of important biological substances that were previously difficult to obtain.

1. RECOMBINANT DNA TECHNOLOGY
Dr. LEKSHMI PRIYA J S
GENETIC ENGINEERING
OR
GENE CLONING
OR
GENE MANIPULATION

2. SPECIFIC LEARNING OBJECTIVES
• History
• What is biotechnology?
• What is genetic recombination ?
• Definn of recombinant DNA & recombinant
DNA technology
• Basic principle
• Steps involved
• Applications
• Limitations

3. HISTORY
• Herbert Boyer (1936-) and Stanley N. Cohen
(1935-) develop recombinant DNA technology
IN 1973, showing that genetically engineered
DNA molecules may be cloned in foreign cells .

4. • Biotechnology may be defined as “the method
by which a living organism or its parts are
used to change or to incorporate a
particular character to another living
organism”
• It involves the application of scientific principles
to the processing of materials by biological
agents.

5. • Genetic recombination is the exchange of
information between two DNA segments.
• This is a common occurrence within the same
species.
• But by artificial means, when a gene of one
species in transferred to another living organism,
it is called recombinant DNA technology.
• In common, this is known as genetic
engineering.

6. Definition of recombinant DNA
• Production of a unique DNA molecule by joining
together two or more DNA fragments not normally
associated with each other.
• DNA fragments are usually derived from different
biological sources .
• MANIPULATION OF DNA TO ACHIEVE A
DESIRED GOAL IN A PREDETERMINED WAY .

7. Definition of recombinant DNA technology
• A series of procedures used to recombine DNA
segments.
• Under certain conditions, a recombinant DNA
molecule can enter a cell and replicate.

8. Basic principle of recombinant DNA technology
• 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 .

10. STEPS
• ISOLATION OF DESIRED DNA
• ISOLATION OF VECTOR
• PRODUCTION OF rDNA/Hybrid DNA/Chimeric DNA
• INTRODUCTION INTO A HOST CELL
• MULTIPLICATION & SELECTION OF CELLS WITH rDNA
MOLECULES
• EXPRESSION OF GENES TO PRODUCE DESIRED
PRODUCT

11. 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.
• DNA exists within the cell membrane along with other
macromolecules such as RNA, polysaccharides, proteins, and lipids.
• 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.

12. CELL
ULTRACENTRIFUGATION
Enzymes , detergent
Phenol
Proteases
Ribonucleases
Chilled ethanol
DNA
Restriction endonucleases, DNA CUTTING ENZYME
Gets seperated by electrophoresis & identified by southern blotting techniques

13. 2. ISOLATION OF VECTOR
Generally prokaryotic vectors are selected because of rapid multiplication
Plasmid  vector in rDNA technology .
BACTERIAL CELL
Enzyme , EDTA, Lysozymes
Sodium lauryl sarcosinate
Plasmid ( sticky end plasmid)
Seperated by ultracentrifugation or electrophoresis

14. 3. FORMATION OF CHIMERIC DNA
Word chimeric DNA is derived from MYTHOLOGY .
Described with narasimha , lions head and mans body
Enzyme  DNA LIGASE
• Needs ATP , because there is formation of phosphodiesterase linkage .
• 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 terminology of genetics this intermixing of different DNA strands
is called recombination.
• Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and
the technology is referred to as the recombinant DNA technology.

15. 4. INTRODUCTION INTO HOST CELL .
5.MULTIPLICATION IN HOST CELL &
SELECTION OF CELLS CONTAINING
CHIMERIC DNA
Done by hybridisation tecnique with the help of probes and or
by using antibiotic registers .
6.Expression of gene to produce desired product .
Produce desired protein on transcription & translation process.

17. Molecular tools in Rdna technology
1.ENZYMES
a)
Exonucleases – cut outer nucleotides .
Endonucleases – act on internal phospho di ester linkage.
Imp enzyme is RESTRICTION ENDONUCLEASE 
Molecular scissor.
Types 1,2 & 3
1 & 3 – requires ATP & cut DNA molecules away from
recognition sequence .

18. • Restriction endonuclease type 2 is important cuts
DNA molecules within a recognition sequence .
• RECOGNITION SEQUENCE  sequence of 4-6
nucleotides which is palindromic,which read similarly
in either directions .
Eg
ECOR1(E coliRy13 I )- Restricion endonuclease Ecoli .
First letter – GENUS
Second letter – SPECIES
Then strain & order of discovery .

19. • DNA Ligase  molecular glue
DNA joining enzyme
requires ATP
causes bond formation;phospho diester
linkage
• Polymerase
• Alkaline phosphatase
• Reverse transcriptase

20. 2. VECTORS transport vehicle
 used for transport of desired piece of
gene into a host cell
1. PLASMID - <10kb
2. BACTERIOPHAGE – 10 -20 kb
3. COSMID – 35-50 kb
4. YEAST ARTIFICIAL CHROMOSOME ( YAC) - 1Mb
5. BACTERIAL ARTIFICIAL CHROMOSOME (BAC) -
<300kb
6. MAMMALIAN ARTIFICIAL CHROMOSOME 1Mb
6 T YPES

21. PLASMIDS
Double stranded
Extra chromosomal material
Independent replication occurs in plasmid
Eg PUC, pBR322

22. 3.Host
Generally prokaryotic hosts are preferred
Bcz of rapid multiplication
Most commonly E Coli & Bacillus subtilis
in eukaryotes – fungi,mammalian cell,plant cell .

23. • Gene transfer techniques
conjugation transformation transduction electroporation
Gene gun
technique
Particle
bombardment

24. Practical Uses of Recombination
• 1. Used to map genes on chromosomes
(recombination frequency proportional to distance
between genes)
• 2. Making transgenic cells and organisms

25. Applications
• Quantitative preparation of biomolecules
• Recombinant Vaccines
• Antenatal diagnosis of genetic diseases
• Monoclonal antibodies
• Cell/tissue culture
• To identify mutations in genes
• Xenotransplantation

26. • To detect activation of oncogenes
• Production of next generation antibiotics
• Genetically modified crops

27. Applications
HUMAN INSULIN
• Insulin is basically a hormone, which is made up of proteins.
• This hormone is secreted in cells of the pancreas that are commonly
referred to as the ‘Islets Of Langerhans’.
• This hormone plays important role in controlling the glucose level in
body, because decreased level of insulin may cause diabetes.
• Recombinant DNA technology has allowed to develop human insulin
by using the bacteria as a host cell.
• A variety of different recombinant insulin preparations are in
widespread use.
• Recombinant insulin is synthesized by inserting the human insulin
gene into E. coli, which then produces insulin for human use.
• This is supposed to be safer than traditionally prepared drugs

28. 1.Obtaining human insulin gene

29. 2.Joining the human insulin gene
into a plasmid vector

30. 3.Introducing DNA plasmids
into bacteria

31. 4.Selecting the bacteria which have
taken up the correct piece of DNA

32. HUMAN GROWTH HORMONES
• Human growth hormone is a polypeptide hormone. It is responsible
for growth, reproduction of the cells and regeneration in humans as
well as animals.
• It is secreted by somatotroph cells present in the pituitary glands.
• Before recombinant HGH became available, HGH for therapeutic use
was obtained from pituitary glands of cadavers. This unsafe practice
led to some patients developing CreutzfeldtJacob disease.
• Recombinant HGH eliminated this problem, and is now used
therapeutically.
• It has also been misused as a performance enhancing drug by athletes
and others.
• In recent days biotechnology has helped scientists to produce many
growth hormones.
• The dwarfism disease is successfully treated with this hormone .

33. • If molecules are isolated from higher organisms, the
availability will be greatly limited.
• For eg.- To get 1 unit of growth hormone, more than 1000
pituitaries from cadavers are required.
• By means of recombinant technology, large scale
availability is now assured

34. • Vaccines
• Vaccine is a biological substance that is prepared from the
suspension of weak or dead pathogenic cells.
• It is injected in the body to enhance the production of antibodies
against a particular antigen.
• Recombinant DNA technology has made it easier for scientists to
develop vaccines by cloning the gene used for protective antigen
protein.
• Viral vaccines are mostly developed from this technique, for
example Herpes, Influenza, Hepatitis, Foot and Mouth disease.

35. • The surface antigen of Plasmodium falciparum, one of
the 4 species of malaria, has been transferred to E. coli
to produce amounts large enough to develop a vaccine
against this form of malaria. It works well enough for
people who will visit a malarious region for a
relatively short period of time.

36. • Is now possible to produce a biological
substance without any contamination.
• Hepatitis, caused by HBV, is highly contagious.
• Absolutely essential to make sure preparations of
vaccines or clotting factors are free from contaminants
such as hepatitis B particles.
• RD-Technology provides the answer to produce safe
antigens for vaccine production

37. • Monoclonal Antibodies
• When a foreign object enters the body, the immune system of the
body produces a specific protein called antibody.
• Hybridoma technique has made it possible to produce
monoclonal antibodies. In this technique, the lymphocytes or B
cells are joined with myeloma cells; the resulting substance is
called as Hybridoma.
• This hybridoma produces unlimited antibodies in culture. The
antibodies produced are called monoclonal antibodies.
• These antibodies are used to produce vaccines against different
viral infections.

38. • Interferon
• A glycoprotein that has the ability to block the multiplication or
division of viruses in the cells or nearby cells are called interferons.
• It can be used to treat cancer like hairy cell leukemia. Recombinant
DNA technology produces this protein using E.coli.
• Interferon alpha is used to treat lymphoma and myelogenous
leukemia.

39. Specific probes for Diagnosis of Diseases
Specific probes are useful for:
i. Antenatal diagnosis of genetic diseases.
For eg.- many of the single gene defects like cystic
fibrosis, phenyl ketonuria etc. Could be identified by
taking cell samples from fetus.
ii. To identify viral particles or bacterial DNA in
suspected blood and tissue samples.

40. iii.To demonstrate virus integration in transformed
cells.
iv. To detect activation of oncogenes in cancer.
v. To pinpoint the location of a gene in a
chromosome.
vi. To identify mutations in genes

41. Gene Therapy
• It is an important applications of RD-Technology .
• Normal genes could be introduced into the patient so
• that genetic diseases can be cured.
• Transferring the gene for normal adult hemoglobin
into marrow stem cells of an individual with sickle-
cell anemia. The goal is to promote the growth of
enough cells to produce enough normal hemoglobin
to alleviate the symptoms of sickle-cell anemia

42. Limitations of Recombinant DNA technology
• Destruction of native species in the environment the genetically
modified species are introduced in.
• Cross contamination and migration of proprietary DNA between
organisms.
• Recombinant organisms contaminating the natural environment.
• The recombinant organisms are population of clones, vulnerable in
exact same ways. A single disease or pest can wipe out the entire
population quickly.
• Creation of superbug is hypothesized.
• . Many people worry about the safety of modifying food and
medicines using recombinant DNA technology

43. Summary

44. To summarise….
• Recombinant proteins are widely used as reagents
in laboratory experiments and to generate
antibody probes for examining protein synthesis
within cells and organisms.
• Thus the use of this advanced technology,
Recombinant DNA technology produces variety
of products which are used for medical purposes.
• It is a challenging field, and play a key role in
preventing genetic diseases, producing targeted
medicines, and providing patients with less toxic
pharmaceuticals.
• Hence it is gaining tremendous significance in the
field of medicine today

45. References
• Textbook of medical biochemistry by D M
Vasudevan & Sreekumari S 8th edition .
• Essentials of biochemistry U satyanarayana

46. THANK YOU