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ENNADA…….RASCALA…MIND IT..!!!!!!
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WILL THERE BE ANOTHER YOU??????............
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YES...!!!!!!!!!!!!
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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.
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DOLLY…. a female domestic sheep, and the first mammal to be cloned from an adult somatic cell
(5 July 1996 – 14 February 2003)
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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 in February 2002.
hhhgenetic
Genetic EngineeringOR
Recombinant DNA Technology
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Introduction
Genetic engineering is
most advanced.
a tool of biotechnology
Sophisticated and
Genetic Engineering includes techniques of DNA analysis
to manipulate DNA
change DNA sequence and bring about a
desirable genetic expression.
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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.
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3. Forensic applications
Medical applications ………contd
4. Production of proteins for
Replacement therapy (e.g. insulin)
Disease prevention (e.g. vaccines)
Diagnostic tests (e.g. monoclonal antibodies).
5. Treatment of genetic diseases (gene therapy)
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.
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.
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.
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Transgenic Goat
Applications in ecology
Recombinant Bacteria- bacteria which can be engineered to “eat” oil spills.
Some Important Tools of Genetic Engineering
1) EnzymesRestriction Endonucleases (REs):DNA ligaseDNA PolymerasesReverse transcriptases
2)VectorsPlasmidBacteriophage, Cosmid Yeast
Some Important Tools of Genetic Engineering
Restriction Endonucleases (REs): used as scissors to cut DNA -DNA scissors
at specific DNA sequences
to generate a set of smaller fragments.
Enzymes
DNA fragments
Genomic DNA
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DNA ligase
Joins two DNA molecules or fragments.
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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
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Vectors
Examples : PlasmidBacteriophage, Cosmid
Yeast
Into the DNA of the vector a foreign DNA can
be inserted, integrated/incorporated.
Use : For amplification by cloning and for
gene therapy.
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Plasmid
present in bacteria
A small, circular, dsDNA
Confer antibiotics resistance against the bacteriamany copies of plasmid in a bacterium
replicate independent of the bacterial DNA.
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Bacteriophage
is a virus that can infect bacteria
Cosmid
plasmid + Cos sites
can carry larger DNA fragments
for binding to bacteriophages
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Restriction Endonucleases (REs)•recognize specific DNA sequences- called
“palindrome” (restriction sites)
•cuts the phosphodiester bonds of the DNA
on both the strands.
• Example : EcoR I (E. coli RY 13) recognises sequence
5’ GAATTC 3’.
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Action of EcoRI
Cuts both strand of the DNA
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Action of RE
PlasmidPlasmid with a cut
RE
DNA REDNA fragment
Restriction enzyme nomenclature
• EcoRI – Escherichia coli strain R, 1st enzyme• BamHI – Bacillus amyloliquefaciens strain H, 1st
enzyme• DpnI – Diplococcus pneumoniae, 1st enzyme • HindIII – Haemophilus influenzae, strain D, 3rd enzyme• BglII – Bacillus globigii, 2nd enzyme• PstI – Providencia stuartii 164, 1st enzyme• Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme• KpnI – Klebsiella pneumoniae, 1st enzyme
Why the funny names?
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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).
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Applications of REs in Genetic Engineering
1) sequencing of DNA
2) cloning of DNA
3) antenatal diagnosis of inherited disorders ( RFLP analysis)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).
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Some Important Techniques in DNA Analysis and Genetic
Engineering:DNA Amplification:
production of many identical copies of a DNA
fragment of interest.
1)further DNA analysis or
2)for large-scale genetic expression
(protein production).
Uses
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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 protein production
shorter time for amplifying DNA fragments
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Cloning
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.
Production of an identical copy of either DNA or a cell or an organism is called cloning.-2 Types.
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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
DNA Cloning
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Chimeric DNA / DNA chimera
Constructing recombinant DNA molecule
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Amplifying the recombinant DNA molecule in a bacterial host
1. Transfection / transformation
2. Amplify in a suitable culture medium
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Selection
Isolation
Amplification
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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
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Applications of recombinant DNA Technology
Used in the fields of
Medicine, Agriculture, Animal
Farming, Ecology, Paleontology, etc.
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Medical applications of Recombinant DNA Technology 1.Production of proteins for
Replacement therapy (e.g. insulin)
Disease prevention (e.g. vaccines)
Diagnostic tests (e.g. monoclonal antibodies).
2.Treatment of genetic diseases (gene therapy)
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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.
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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).
Human insulin is produced using Recombinant DNA Technique :
Recombinant Human Growth Hormone
Recombinant insulin (Humulin)
Vaccines production
• Vaccines like hepatitis B vaccine, are produced using Recombinant DNA Technique
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Polymerase Chain Reaction (PCR) in vitro method for DNA amplification
much faster
more sensitive method than cloning.
can only amplify short segments of DNA
cannot be used for amplifying genes and
for production of proteins
very little DNA sample is sufficient
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Procedure : Use : To amplify a short sequence of DNA
DNA sample + dNTP’s + Primers +Enzyme : Taq DNA polymerase
(2) Treatment of the mixture :
94 - 95 C
52 - 54 C
72 C
Denaturation of DNA 30 – 60 sec
Annealing of primers 30 – 60 sec
Extension of the DNA 1 min
(1) A mixture of
1 cycle
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Primers anneal1 cycle
Product : Every cycle the DNA doublesNo. of cycles : 30 - 45
Separation DNA strands
Extension by DNAP
Test DNA sample
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Applications of PCR
Useful : when insufficient DNA molecules are
present in test samples for DNA
analytical techniques.
1. Very little DNA sample is required
2. Amplification time is very short.
3. Amplification rate is high.
Advantages of PCR
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Uses of PCR1. Diagnostic uses
used to quickly detect microbial infections,
when the number of microbes is less in the
sample.
Examples :Diagnosis of
Tuberculosis (TB)
AIDS
Mycobacterium tuberculi
HIV
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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)
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3. Forensic Uses:
Samples used : Blood, saliva, semen, hair
Obtained from : a victim or suspect
Volume of the sample : is insufficient
Sample Amplification of DNAPCR
Amplified DNA DNA analytical techniquesi.e., DNA fingerprinting
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Gene therapy : treatment of Genetic disorders .
•Severe Combined Immuno Deficiency (SCID)
• Cystic Fibrosis
• Familial Hypercholesterolemia
• Hemophilia
Adenosine deaminase
Chloride channel
Receptor for LDL
Clotting factor (factor VIII or IX)
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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.
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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.
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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.
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Isolation Of The Healthy Gene
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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.
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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.
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Replacement of mutant protein in genetic disorders
Major vectors used for transfer of the gene in gene therapy
Retroviruses
Adenoviruses
Plasmid-liposome complexes.
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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.
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o Incorporation into a vector - DNA Chimera
o Cloned
o Protein expressed Suitable bacteria
1. For Protein expression Uses of DNA library
2. As a probe for analytical techniques
o Detecting specific nucleotide sequence in
test samples.o As in Southern and Northern blot techniques.
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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
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Examples for probes
RNA
Synthetic oligonucleotides
Antibodies (protein) -as a probe for protein
molecule -in Western blot technique.
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Blot techniques :
Southern
Northern
Western
Types
DNA
RNA
Protein
Detection of
Analytical techniques used in Recombinant technology
Done on test samples
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Applications of Blot techniques : in
• Research
• Diagnosis of diseases (microbial and genetic)
• Forensic medicine.
Southern Blot TechniqueProcess : 6 steps
1) Extraction of DNA from the test sample/cells
2) Digestion by a suitable RE – Product DNA fragments
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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.
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Blood stain
DNA extracted RE treated DNA fragments separated
by gel electrophoresis
DNA denaturation with alkali
Nitrocellulose membrane
Transfer to membrane
Exposure to X-ray film
DNA Probes (radiolabeled) to the membrane
DNA pattern
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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.
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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.
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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.
Medical Applications of Genetic Engineering
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• 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
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Sample Amplification of DNAPCR
Amplified DNA DNA analytical techniques
3. Forensic Uses:
For identifying dead bodies
Settling parental disputes.
Identifying criminals.
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Samples used : Blood, saliva, semen, hair
Obtained from : a victim or suspect
Volume of the sample : is insufficient
Sample Amplification of DNAPCR
Amplified DNA DNA analytical techniquesi.e., DNA fingerprinting
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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.
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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).
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5. Treatment of genetic diseases :
Example : Gene therapyInvolves
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.
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Genetic disorders treated by Gene therapy (attempt) :
•Severe Combined Immuno Deficiency (SCID)
• Cystic Fibrosis
• Familial Hypercholesterolemia
• Hemophilia
Adenosine deaminase
Chloride channel
Receptor for LDL
Clotting factor (factor VIII or IX)
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1. What are restriction Endonucleases? Give two examples. (3)2. What is reverse transcriptase? What is its significance? (3)3. Reverse transcriptase. (3)4. 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)
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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.
MULTIPLE CHOICE QUESTIONS
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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.
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5. ____________ is an example for a recombinant
protein used in disease prevention.
a) HB antigen b) Interleukins
c) Interferons d ) Insulin.
.6. ___________ is an example for a recombinant
protein used in replacement therapy.
a) HB antigen b) Antibodies
c) Oral polio vaccine d ) Insulin.
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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.
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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.
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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.
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RNA cDNA dsDNA
