Basic principles of genetic engineering. Study of cloning vectors, restriction endonucleases and DNA ligase. Recombinant DNA technology. Application of genetic engineering in medicine. Application of r DNA technology and genetic engineering in the products: a. Interferon b. Vaccines- hepatitis- B c. Hormones- Insulin. Polymerase chain reaction Brief introduction to PCR Basic principles of PCR

1. GENETIC ENGINEERING
Principle, Tools, Techniques,
Types and Application
Presented by:
Mr. TARUN KAPOOR
Assistant Professor,
Shri Ram College of Pharmacy, Karnal

2. Contents
• Genetic Engineering: An Introduction & Basic principles
• Tools of Genetic Engineering (Enzymes and transformation factors)
Cloning vectors, restriction endonucleases and DNA ligase
• Methods of Gene transfer
• Methods of DNA amplification (Gene Cloning and PCR)
• Recombinant DNA technology & its Application
• Recombinant Pharmaceuticals: An Overview
• Production of genetic engineering products (Interferon, Vaccines-
hepatitis- B, Insulin)
• Brief introduction to PCR: Principle, Advantages and Applications

3. Genetic Engineering
• Genetic Engineering involves manipulation of genetic
material, is also called Recombinant DNA technology or
Gene Cloning.
• Genetic recombination technology consists of the breakage
and joining of DNA molecules.
• Genetic engineering primarily involves the manipulation of
genetic material ( DNA) to achieve the desire goal in pre
determined way.
• Other terms are Recombinant DNA technology, Gene
manipulation, Gene cloning, Genetic modifications.

4. Basic principle of Genetic
Engineering
• DNA fragment of interest is obtained by cleaving
chromosomes by Restriction endonuclease.
• Cloning vector is cleaved with Restriction
endonuclease.
• Fragments are ligated to the prepared cloning vector.
• Recombinant vector DNA is introduced into the host
cell
• Propagation (cloning) produces many copies of
recombinant DNA
• The gene is extracted and harvested the product

7. Tools of Genetic Engineering
1) Enzymes
 Restriction Endonucleases (REs)
 DNA ligase
 DNA Polymerases
 Reverse transcriptase
2) Transformation factors
 Plasmid
 Bacteriophage
 Cosmid
 Yeast

8. Restriction Endonucleases (REs)
 Used as mol. scissors to cut DNA –DNA at specific
DNA sequences
 To generate a set of smaller fragments
 Recognize specific DNA sequences called
“palindrome” (restriction sites) Example : EcoRI
recognizes sequence cuts the Phosphodiesterase
bonds of the DNA on both the strands

9. Plasmid Plasmid with a cut
RE
DNA
RE
9
DNA
fragment

11. Applications of RE
1) Sequencing of DNA
2) Cloning of DNA
3) Antenatal diagnosis of inherited disorders
4) DNA finger printing (having forensic applications)
5) For Southern blot technique (for detecting the
presence of a particular base sequence in the sample
DNA)

12.  DNA ligase
– Joins two DNA molecules or fragments.
 DNA Polymerases
– Synthesis of DNA using DNA template
 Reverse transcriptase
– Enzyme found in retroviruses that makes
DNA copy, using RNA as template

13. Transformation factors
• Into the DNA of the vector a foreign DNA can be
inserted, integrated/incorporated.
• Use : For amplification by cloning and for gene
therapy.
• Plasmid, Bacteriophage, Cosmid, Yeast

14. Cloning Vectors
• Vectors are the DNA molecule, which can carry A
foreign DNA fragment to be cloned. The are self
replicating in an appropriate host cell.
• The most important vectors are Plasmids,
Bacteriophages & Cosmid.
• An ideal characteristics of an vector is should be
small in size with endonuclease site.

15. Plasmid
 A small, circular, dsDNA present in bacteria
 Confer antibiotics resistance against the bacteria
 Many copies of plasmid in a bacterium
 Replicate independent of the bacterial DNA.

16. Cosmid: Cosmid are vectors posses the characteristic of
both plasmid and bacteriophage.
 Can carry larger DNA fragments
 Cosmid can be constructed by adding a fragment of
DNA to plasmid.

17. • Yeast Artificial Chromosomes (YAC) is a synthetic
DNA that can accept large fragment (particular human
DNA). It is possible to clone large DNA pieces by
using YAC.
• Bacteriophage: It is a virus that can infect bacteria.

18. Methods of gene Transfer
• Transformation
• Transduction
• Electroporation
• Conjugation
• Microinjection
• Liposome mediated
gene transfer

19. Transformation

20. • Electroporation: Application of high voltage electrical field
to cells
• Direct transfer:
– Micro injection
– Particle bombardement
• Liposome mediated gene transfer

22. Types of DNA amplification
 Cloning
• In vivo method using bacteria
• Used to amplify longer segments of DNA
• Suitable for large scale protein production
 Polymerase Chain Reaction (PCR)
• In vitro method using DNA polymerase
• Shorter segments of DNA can be amplified
• Shorter time for amplifying DNA fragments

23. DNAAmplification
• Production of many identical copies of a DNA
fragment of interest.
• Uses
1) Further DNA analysis
2) For large-scale genetic expression
3) Protein production

24. Cloning
• Production of an identical copy of either DNA or a cell
or an organism is called cloning.
• It is of 2 Types:
1. Molecular cloning: Production of identical DNA
molecules (i.e., identical in base-sequence)
2. Somatic cloning: Production of cells or
organisms with identical genetic makeup

25. • Genetic recombination is exchange of information between
two DNA segments within same species. But artificially
when a gene of one species is transferred to another living
organism, it is called recombinant DNA technology or
genetic engineering.
• rDNA: Production of a unique DNA molecule by joining
together two or more DNA fragments which are derived
from different biological sources.
• rDNA technology: A series of procedures used to
recombine DNA segments. Under certain conditions, a
recombinant DNA molecule can enter into cell & replicate.

26. History of rDNA technology
• Recombinant DNA technology is one of the
recent advances in biotechnology, which was
developed by two scientists named Boyer and
Cohen in 1973.

27. Applications of rDNA Technology
1. Large scale production of human proteins by genetically
engineered bacteria.
–Recombinant human insulin
–Recombinant human growth hormone
–Recombinant blood clotting factors
–Recombinant hepatitis B vaccine
–Cytokines and growth factors (IF, IL)
–Monoclonal antibodies
–Recombinant enzymes
–Recombinant HIV protein for ELISA testing
–Albumin, fibrinolytic and thrombolytic agents

29. 2) Basic research – understanding structure and functions of
DNA & proteins (Human Genome Project)
3) Gene therapy for genetic diseases
4) Food production
5) Plant: Genetically modified corn
6) Forensic applications
7) Genetically modified organisms are called transgenic
organisms. Mice (Study human immunity), Chicken
(Resistant to infection), Cows (Increase milk & leaner
meat)
8) Applications in ecology: Recombinant Bacteria which can
be engineered to “eat” oil spills.

30. Recombinant Pharmaceuticals
• Human Insulin
• Human Growth Hormone
• Human blood clotting factors
• Vaccines
• Monoclonal Antibodies
• Interferons
• Antibiotics & other secondary metabolites

31. Human Insulin
• Insulin is a hormone produced by β-islets of Langerhans of
pancreas. It was discovered by sir Edward Sharpey Schafer
(1916) while studying Islets of Langerhans.
• People who do not produce the necessary amount of insulin
have diabetes.
• Chemically, insulin is protein consist of 51 amino acids, 30
construct polypeptide chain B and 21 amino acids construct
polypeptide chain A and both chains linked by disulfide
bond.

33. • Modify E.coli cells to produce insulin; performed by Genentech
in 1978
• Prior, bovine and porcine insulin used but induced immunogenic
reactions. Also, there were many purification and contamination
hassles. To overcome these problems, researchers inserted human
insulin genes into a suitable vector (E.coli).
• First, scientists synthesized genes for the two insulin A & B
chains. Then inserted into plasmids. The genes were inserted in
such a way that the insulin & B-galactosidase residues would be
separated by a methionine residue. This is so that the insulin A &
B chains can be separated easily by adding cyanogen bromide.

34. Producing Recombinant Insulin
• The vector was then transformed into E.coli cells.
• Once inside the bacteria, the genes were
"switched-on" by the bacteria to translate the
code into either the "A" chain or the "B" chain
proteins found in insulin
• The purified insulin A and B chains were then
attached to each other by disulphide bond
formation under laboratory conditions

36. Hepatitis B Vaccine
• Hepatitis B virus (HBV) is common infectious diseases. WHO
estimates that there are 285 million chronic carriers of HBV
worldwide.
• Hepatitis B is 50 to 100 times more infectious than AIDS. Hepatitis B
is irritation and swelling (inflammation) of liver due to infection with
hepatitis B virus (HBV). Other types include: Hepatitis A, C and D.
• It produces several chronic liver disorders such as Liver cirrhosis and
primary liver cancer.
• Hepatitis B Recombinant Vaccine: It’s a novel and significant
developed vaccine which is produced from the antigenic proteins of
Hepatitis B virus by recombinant process that duplicates the chemical
messages and secreted factors (Interleukin-2) for the communication
and activity of immune cells.

37. Production of recombinant HBV
Vaccine
Production of these genes is needed in order to get production of
vaccines on a large scale. A general procedure for the production of
recombinant Hepatitis B vaccines are described here-
1. HBs antigen producing gene is isolated from HB virus by isolation
process (cell lysis, protein denaturation, precipitation,
centrifugation and drying).
2. A plasmid DNA is extracted from a bacterium- E.coli and is cut
with restriction enzyme- Eco RI forming the plasmid vector.
3. The isolated HBs antigen producing gene is located and inserted
into the bacterial plasmid vector on forming the recombinant
DNA.

38. 4. This recombinant DNA, containing the target gene, is
introduced into a yeast cell forming the recombinant yeast
cell.
5. The recombinant yeast cell multiplies in the fermentation
tank and produces the HBs antigens.
6. After 48 hours, yeast cells are ruptured to free HBsAg. The
mixture is processed for extraction.
7. The HBs antigens are purified. HBsAg are combined with
preserving agent and other ingredients and bottled.
8. Now it is ready for vaccination in humans.

40. INTERFERONS
Interferons (IFNs) were the first family of cytokines to be discovered.
In 1957 researchers observed that if susceptible animal cells were
exposed to a colonizing virus, these cells immediately become
resistant to attack by other viruses. This resistance was induced by a
substance secreted by virally-infected cells, which was named
‘interferon’ (IFN). Humans produce at least three distinct classes,
IFN-a, IFN-b and IFN-g.
Biological effects: Induction of cellular resistance to viral attack.
Regulation of most aspects of immune function. Regulation of
growth and differentiation of many cell types. Sustenance of early
phases of pregnancy in some animal species.

41. Production of Interferons
• A DNA sequence coding for the product was synthesized and
inserted into E. coli. The recombinant product accumulates
intracellularly as inclusion bodies.
• Large-scale manufacture entails an initial fermentation step. After
harvest, the E. coli cells are homogenized and the inclusion bodies
recovered via centrifugation.
• After solubilization and refolding, the interferon is purified to
homogeneity by a combination of chromatographic steps.
• The final product is formulated in the presence of a phosphate
buffer and sodium chloride.
• It is presented as a 30 mg/ml solution in glass vials and displays a
shelf- life of 24 months when stored at2–8°C.

42. Human
Fibroblast
Human Interferon
β Gene
Modified Human Interferon
β Gene
Plasmid
Restriction enzyme cut
Plasmid
E. Coli containing its Own
gene
E. Coli
containing
rDNA
Replicated E.
Coli
producing
IFN β
Purified
Interferon
1β
Packed and
Ready for Use
rDNA

44. Polymerase Chain Reaction
• PCR is an in vitro technique for the amplification of DNA.
• Developed by Kary Mullis in the 1980s
• Much faster
• More sensitivemethod than cloning.
• Very little DNA sample is sufficient
• Can only amplify short segments of DNA
• Cannot be used for amplifying genes and for production of
proteins

45. Principle
• Double stranded DNA of interest is denatured to
separate strands
• Each strand is then allowed to hybridize with a
primer. The primer template duplex is used for
synthesis.
• Program thermocycler for times include three steps:
Denaturation, anneling and extension repeated again
and again to generate multiple forms of target DNA.
• The primer extension product synthesized in 1 cycle
serve as template for next cycle.

46. Procedure
• A mixture of DNA sample + dNTP + Primer + Enzyme
: Taq DNA polymerase
• Treatment of the mixture :1 cycle
 94 – 95˚ C : Denaturation of DNA : 30 – 60 sec
 52 – 54˚ C : Annealing of primers : 30 – 60 sec
 72˚ C : Extension of the DNA : 1 min

48. Types of PCR:
– Real-time PCR
– Nested PCR
– Inverse PCR
– Reverse transcription PCR
Advantages of PCR
1. Very little DNA sample is required
2. Amplification time is very short.
3. Amplification rate is high.
Uses: When insufficient DNA molecules are present in test
samples for DNA analytical techniques.

49. Applications of PCR
1. Diagnosis: Bacterial and viral diseases (TB, Hepatitis C, HIV)
2. Medicolegal/ Forensic cases: DNA amplification from hair,
saliva, semen and blood sample (DNA fingerprinting)
3. Diagnosis of genetic disorders: SCD, thalassemia, cystic fibrosis
4. Prenatal diagnosis of inherited disorders
5. Cancer detection:
i. To monitor abnormal cells present in treated patients.
ii. Identification of mutation in oncosuppressor
genes
5. Fossil studies: To study evolution by comparing the sequences in
the extinct and living organism