Genetic engineering,recombinant DNA technology..

1. 1

2. ENNADA…….RASCALA…MIND IT..!!!!!!
2

3. WILL THERE BE ANOTHER
YOU??????............
3

4. YES...!!!!!!!!!!!!
4

5. Fifteen years ago, scientists in
Edinburgh announced to the world an
incredible breakthrough: the creation
of the first cloned animal–a sheep who
originated from a cell taken from an
adult mammal.
Dolly’s birth sparked a vigorous debate
about the controversial technique and
its potential application to humans.
5

6. DOLLY….  a female domestic sheep, and
the first mammal to be cloned from an
adult somatic cell
(5 July 1996 – 14 February 2003)
6

7. Copycat….!!!!!!!!!!!!!
The world's first cloned kitten, named Cc. It was created
by scientists in Texas using a cell taken from an adult
tortoise shell. The photo, taken on December 22 2001
when the kitten was seven weeks old, was made public
7
in February 2002.

8. Genetic Engineering
OR
Recombinant DNA
hhhgenetic
Technology

9. Introduction
Genetic engineering is a tool of biotechnology
Sophisticated and most advanced.
Genetic Engineering includes techniques

of DNA analysis

to manipulate DNA
change DNA sequence and bring about a
desirable genetic expression.
9

10. Applications of Genetic engineering
fields of medicine, agriculture, animal
farming, ecology, paleontology, etc.
Medical applications of DNA technology
1. Basic research - understanding of
structure and functions of DNA & proteins.
2. Diagnosis of diseases - genetic and microbial.
10

11. Medical applications ………contd
3. Forensic applications
4. Production of proteins for
Replacement therapy
Disease prevention
(e.g. insulin)
(e.g. vaccines)
Diagnostic tests (e.g. monoclonal antibodies).
5. Treatment of genetic diseases (gene therapy)
11

12. Applications in agriculture
PLANTS
1. disease-resistant and
insect-resistant, high
yielding crops
2. Hardier fruit
3. 70-75% of food in
supermarket is
genetically modified.

13. Applications in animal farming
Genetically modified
organisms are called
transgenic organisms.
1.
Mice – used to study human
immune system
2.
Chickens – more resistant to
infections
3.
Cows – increase milk supply
and leaner meat
4. Goats, sheep and pigs –
produce human proteins in
their milk.

14. Transgenic Goat
Human DNA in
a Goat Cell
.
This goat contains a human
gene that codes for a blood
clotting agent. The blood
clotting agent can be harvested
in the goat’s milk.

15. Applications in ecology
Recombinant Bacteria- bacteria which can be
engineered to “eat” oil spills.

16. Some Important Tools of Genetic Engineering
1) Enzymes
Restriction Endonucleases (REs):
DNA ligase
DNA Polymerases
Reverse transcriptases
2)Vectors
Plasmid
Bacteriophage,
Cosmid
 Yeast

17. Some Important Tools of Genetic Engineering
Enzymes

Restriction Endonucleases (REs):

used as scissors to cut DNA -DNA scissors

at specific DNA sequences

to generate a set of smaller fragments.
Genomic
DNA
DNA
fragments
17

18.  DNA ligase
Joins two DNA molecules or fragments.
18

19.  DNA Polymerases
Synthesis of DNA using DNA template and
dNTPs
 Reverse transcriptase
Enzyme found in retroviruses that makes
DNA copy, using RNA as template
RNA
cDNA
dsDNA
19

20. Vectors
Into the DNA of the vector a foreign DNA can
be inserted, integrated/incorporated.
Use : For amplification by cloning and for
gene therapy.
Examples : Plasmid
Bacteriophage,
Cosmid
Yeast
20

21.  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.
21

22.  Bacteriophage
is a virus that can infect bacteria
 Cosmid
plasmid + Cos sites
for binding to bacteriophages
can carry larger DNA fragments
22

23. Restriction Endonucleases (REs)
•recognize specific DNA sequences- called
“palindrome”
(restriction sites)
• Example : EcoR I (E. coli RY 13) recognises sequence
5’ GAATTC 3’.
•cuts the phosphodiester bonds of the DNA
on both the strands.
23

24. Action of EcoRI
Cuts both strand of the DNA
24

25. Action of RE
Plasmid
RE
DNA
RE
Plasmid
with a cut
DNA
fragment
25

26. Restriction enzyme nomenclature
Why the funny names?
•
•
•
•
•
•
•
•
EcoRI –
BamHI –
DpnI –
HindIII –
BglII –
PstI –
Sau3AI –
KpnI –
Escherichia coli strain R, 1st enzyme
Bacillus amyloliquefaciens strain H, 1st enzyme
Diplococcus pneumoniae, 1st enzyme
Haemophilus influenzae, strain D, 3rd enzyme
Bacillus globigii, 2nd enzyme
Providencia stuartii 164, 1st enzyme
Staphylococcus aureus strain 3A, 1st enzyme
Klebsiella pneumoniae, 1st enzyme

27. Restriction Endonucleases (REs)
•Examples:
EcoRI;
Hpa I;
BamHI;
Taq I.
REs are isolated from bacteria.
Biological function of RE in bacteria :
is to recognize and cleave foreign DNA
(e.g. DNA of an infecting virus).
27

28. Applications of REs in Genetic Engineering
1)
sequencing of DNA
2)
cloning of DNA
3)
4)
antenatal diagnosis of inherited disorders
( RFLP analysis)
DNA finger printing
(having forensic applications)
5)
for Southern blot technique
(for detecting the presence of a particular
base sequence in the sample DNA).
28

29. Some Important Techniques in DNA Analysis
and Genetic Engineering:
DNA Amplification:
production of many identical copies of a DNA
fragment of interest.
Uses
1) further DNA analysis or
2) for large-scale genetic expression
(protein production).
29

30. Types of DNA amplification
Cloning
 Polymerase Chain Reaction
(PCR)
in vivo method
using bacteria
an in vitro method using
DNA polymerase
used to amplify
longer segments of
DNA
shorter segments of DNA
can be amplified
suitable for large-scale shorter time for amplifying
DNA fragments
protein production
30

31. Cloning
Production of an identical copy of
either DNA or a cell or an organism
is called cloning.
-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.
31

32. DNA Cloning
Recombinant DNA Technology- Cloning a DNA
Fragment
Two principal steps :
 Constructing a recombinant DNA molecule
-gene of one species is transferred to another
living organism.
-usually, a human gene is transferred to a
bacteria.
 Amplifying the recombinant DNA
molecule in a bacterial host
32

33. Constructing recombinant DNA molecule
Chimeric DNA
/ DNA chimera
33

34. Amplifying the recombinant DNA molecule
in a bacterial host
1. Transfection /
transformation
2. Amplify in a suitable
culture medium
34

35. Selection
Isolation
Amplification
35

36. 2. Selection, Isolation and Amplification of
Recombinant DNA:

by specific techniques

(eg. by antibiotic sensitivity technique)

and allowed to multiply in a suitable culture.
3. Release of the Cloned DNA Molecules from
the Bacteria:
by using the same RE as used for cleaving of DNA
36

37. Applications of recombinant DNA
Technology
Used in the fields of
Medicine, Agriculture, Animal
Farming, Ecology, Paleontology, etc.
37

38. Medical applications of Recombinant DNA
Technology
1.Production of proteins for
Replacement therapy
Disease prevention
(e.g. insulin)
(e.g. vaccines)
Diagnostic tests (e.g. monoclonal antibodies).
2.Treatment of genetic diseases (gene
therapy)
38

39. Production of Proteins Using Recombinant
DNA Technique :

proteins, especially human proteins

produce large amounts of proteins
 provide human proteins, which are not
antigenic when administered to humans.
39

40. Proteins produced are used for:
 Replacement therapy and other treatments
(e.g. insulin, growth
hormone, interleukins, antihemophilic
 factor, interferon, etc.).
Disease prevention
(e.g. vaccines, such as hepatitis B antigen)
 Diagnostic tests
(e.g. monoclonal antibodies).
40

41. Human insulin is
produced using
Recombinant DNA
Technique :
Recombinant insulin (Humulin)
Recombinant Human Growth Hormone

42. Vaccines production
• Vaccines like hepatitis B
vaccine, are produced using
Recombinant DNA Technique

43. Polymerase Chain Reaction (PCR)
 in vitro method for DNA amplification
 much faster
 more sensitive method 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
43

44. Use : To amplify a short sequence of DNA
Procedure :
(1)
A mixture of
DNA sample + dNTP’s + Primers +
Enzyme : Taq DNA polymerase
(2)
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
44

45. Test DNA sample
Separation DNA strands
Primers anneal
1 cycle
Extension by DNAP
No. of cycles : 30 - 45
Product : Every cycle the DNA doubles
45

46. Advantages of PCR
1. Very little DNA sample is required
2. Amplification time is very short.
3. Amplification rate is high.
Applications of PCR
Useful : when insufficient DNA molecules are
present
in
test
samples
for
DNA
analytical techniques.
46

47. Uses of PCR
1. Diagnostic uses
used to quickly detect microbial
infections, when the number of microbes is
less in the sample.
Examples :Diagnosis of
Tuberculosis (TB) Mycobacterium tuberculi
AIDS
HIV
47

48. 2. Prenatal diagnosis of genetic disorders
Sections of genes, having particular
mutations known to cause a disease are
 Amplified
 Sequenced
 Diagnosis
Example : Detection of
Sickle cell anemia (HbS)
48

49. 3. Forensic Uses:
Samples used : Blood, saliva, semen, hair
Obtained from : a victim or suspect
Volume of the sample : is insufficient
Sample
PCR
Amplified DNA
Amplification of DNA
DNA analytical
techniques
i.e., DNA fingerprinting
49

50. Gene therapy : treatment of Genetic disorders .
•Severe Combined Immuno Deficiency (SCID)
Adenosine deaminase
• Cystic Fibrosis
Chloride channel
• Familial Hypercholesterolemia
Receptor for LDL
• Hemophilia
Clotting factor (factor VIII or IX)
50

51. Gene therapy
• Gene therapy is aimed at treating genetic
disorders
• involves introduction of normal foreign gene
into somatic cells of the patient having the
genetic disease to compensate for the defective
protein, which is the product of the mutant
gene.
51

52. Gene therapy-procedure
• The procedure involves,
1) isolation of the healthy gene
2) incorporation of this gene into a carrier or
vector and
3) delivering the vector into the target cells.
52

53. Gene therapy-proceedure
• isolation of the healthy gene
-done by isolating its mRNA first
-using this mRNA as a template, cDNA is
synthesised, using reverse transcriptase.
-from thic cDNA, double strande DNA is
synthesised using DNAP.
53

54. Isolation Of The Healthy Gene
54

55. Gene therapy-proceedure..
2) incorporation
of this gene into a
carrier or vector
-Vectors used are
retroviruses, adenoviruses and
plasmid-liposome complexes.
-this is done by recombinant DNA
technology, as described earlier.
55

56. Gene therapy-proceedure….
• delivering the vector into the target
cells.
• The vector with normal gene is now introduced
to the patient.
•The cells of this patient will start producing the
normal protein which was deficient earlier.
•
Disease is cured.
56

57. Replacement of mutant
protein in genetic disorders
57

58. Major vectors used for transfer of the gene
in gene therapy

Retroviruses

Adenoviruses

Plasmid-liposome complexes.
58

59. Reverse Transcriptase (RT)
RNA
cDNA
dsDNA
stored in DNA library
DNA library
a collection of DNA fragments of one
organism, each carried by a plasmid / virus.
59

60. Uses of DNA library
1. For Protein expression
o
Incorporation into a vector - DNA Chimera
o Cloned
Suitable bacteria
o Protein expressed
2. As a probe for analytical techniques
o Detecting specific nucleotide sequence in
test samples.
o As in Southern and Northern blot techniques.
60

61. DNA Probes are
Single stranded, fragments / pieces of DNA
Contain nucleotide sequence complimentary to
the target sequence
Radiolabeled with radioisotopes (usually 32P)
to visualize on an X-ray film
Use : for detecting a target sequence in
Southern and Northern blot techniques
61

62. Examples for probes
 Synthetic oligonucleotides
 RNA
 Antibodies (protein) -as a probe for protein
molecule -in Western blot technique.
62

63. Blot techniques :
Analytical techniques used in
Recombinant technology
Done on test samples
Types
Detection of
 Southern
DNA
 Northern
RNA
 Western
Protein
63

64. Applications of Blot techniques : in
• Research
• Diagnosis of diseases (microbial and genetic)
• Forensic medicine.
Southern Blot Technique
Process
:
6 steps
1) Extraction of DNA from the test sample/cells
2) Digestion by a suitable RE
– Product DNA fragments
64

65. 3) Electrophoresis of the digest
- Separation of fragments
4) Denaturation of DNA and blotting onto a
membrane (nitrocellulose membrane)
5) Adding a radiolabeled DNA probe
6) Autoradiography : Visualization on X-ray film .
DNA fragments hybridized with the
radiolabeled DNA probes.
65

66. Blood
stain
DNA
extracted
Transfer to
membrane
RE treated
Nitrocellulose
membrane
DNA Probes
(radiolabeled) to the
membrane
Exposure to
X-ray film
DNA fragments separated
by gel electrophoresis
DNA denaturation
with alkali
DNA pattern
66

67. The pattern observed on Southern blot
analysis depends on :
•
the specific RE used
•
location of the restriction site in
the DNA sample
• the probe used.
67

68. Restriction Fragment Length Polymorphism (RFLP)
Analysis
 Extraction of Human chromosome

Digestion with one or more REs

Southern blot

Visualization
Application :
molecular analysis of genes involved in disease.
68

69. Medical Applications of Genetic Engineering
1. Basic research for understanding structure
and functions of DNA and proteins.
Recombinant DNA technology has made possible :
• Complete sequencing of the human genome
(Human Genome Project)
• Gene localizing and
• defining the map of the human genome.
69

70. • Isolation and detailed molecular analysis of
genes
involved
in
disease
(using
RFLP
analysis).
2. Diagnosis of diseases - genetic and microbial.
Techniques used : PCR, Southern blot & RFLP
Test sample
: Amniotic fluid
Time of Test
: Prenatal diagnosis
70

71. Sample
PCR
Amplified DNA
Amplification of DNA
DNA analytical
techniques
3. Forensic Uses:

For identifying dead bodies

Settling parental disputes.

Identifying criminals.
71

72. Samples used : Blood, saliva, semen, hair
Obtained from
: a victim or suspect
Volume of the sample : is insufficient
Sample
PCR
Amplified DNA
Amplification of DNA
DNA analytical
techniques
i.e., DNA fingerprinting
72

73. 4. Production of Proteins Using Recombinant
DNA Technique :

proteins, especially human proteins

produce large amounts of proteins
 provide human proteins, which are not
antigenic when administered to humans.
73

74. Proteins produced are used for:
 Replacement therapy and other treatments
(e.g. insulin, growth
hormone, interleukins, antihemophilic
 factor, interferon, etc.).
Disease prevention
(e.g. vaccines, such as hepatitis B antigen)
 Diagnostic tests
(e.g. monoclonal antibodies).
74

75. 5. Treatment of genetic diseases :
Example :
Gene therapy
Involves

Introduction of normal foreign gene

Into somatic cells of the patient having the
genetic disease

To compensate for the defective protein,

Which is the product of the mutant gene.
75

76. Genetic disorders treated by Gene therapy
(attempt) :
•Severe Combined Immuno Deficiency (SCID)
Adenosine deaminase
• Cystic Fibrosis
Chloride channel
• Familial Hypercholesterolemia
Receptor for LDL
• Hemophilia
Clotting factor (factor VIII or IX)
76

77. 1.
2.
3.
4.
What are restriction Endonucleases? Give two examples.
(3)
What is reverse transcriptase? What is its significance?
(3)
Reverse transcriptase.
(3)
What is plasmid? What are its applications in recombinant DNA
technology?
(4)
5. Discuss in detail recombinant DNA technology and its clinical
application.
(5)
6. What is “Recombinant DNA”? Mention applications of genetic
engineering.
(1+3 =4 )
7. Describe the clinical applications of recombinant DNA technology. (4 )
8. Give two applications of recombinant DNA technology.
(3)
9. What is Polymerase Chain Reaction (PCR)? Mention application of
PCR.
(3)
10.What is polymerase chain reaction? Mention its applications
(3)
11.PCR
(4 )
12.Polymerase chain reaction
(3)
13.Gene therapy
(4)
14.What is gene therapy? Name vectors used for gene therapy. (3)
77

78. MULTIPLE CHOICE QUESTIONS
1. DNA Scissors is______________.
a) DNA Polymerase
b) 3’→5’ Exonuclease
c) Restriction endonuclease d) RNase H.
2. Two fragments of DNA are joined by ______.
a) DNA Polymerase b) DNA Ligase
c) Topoisomerase
d) Reverse transcriptase.
78

79. 3. An example for an RNA dependant DNA
polymerase is ____________.
a) DNA Polymerase b) RNA Polymerase
c) Primase
d ) Reverse transcriptase.
4. ____________ confer antibiotic resistance to
bacteria.
a) Genomic DNA
b) Mitochondria
c) Cell wall
d ) Plasmids.
79

80. 5. ____________ is an example for a recombinant
protein used in disease prevention.
a) HB antigen
b) Interleukins
c) Interferons
d ) Insulin.
. ___________ is an example for a recombinant
6.
protein used in replacement therapy.
a) HB antigen
b) Antibodies
c) Oral polio vaccine
d ) Insulin.
80

81. 7. Applications of REs in genetic engineering include
these except_____________.
a) Cloning of DNA
b) Antenatal diagnosis of inherited disorders
c) Radiolabeling
d) DNA finger printing. .
8. The function of polymerase chain reaction is to
_____________.
a) amplify DNA
b) destroy DNA
c) synthesize proteins d) confer antibiotic resistance.
81

82. 9. These can be used as a vector in DNA cloning
except
a) Plasmid
b) Cosmid
c) Oligonucleotides
d) Bacteriophage.
10. Inserting DNA fragment of interest into the DNA
of. a vector produces a molecule which is called by all
these names except
a) Recombinant DNA
b) Recombinant protein
c) DNA chimera
d) Chimeric DNA.
82

83. 11. Ideally, for cloning, both the vector and the DNA
of interest should be cleaved with the same
a) Endonuclease
b) Exonuclease
c) Restricted endonuclease d) RNase H.
12. These are advantages of PCR technique except
a) High rate of amplification
b) Less time required
c) Small amounts of test sample is needed
d) High rate of errors.
83

84. RNA
cDNA
dsDNA
84