recombinant dna technology part 1
TRANSCRIPT
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WILL THERE BE ANOTHER YOU??????............
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YES...!!!!!!!!!!!!
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• Twenty 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.
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hhhgenetic
Genetic EngineeringOR
Recombinant DNA Technology
Dr. Gangadhar ChatterjeeMBBS;MD
Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India
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IntroductionGenetic 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.7
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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)
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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.
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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.
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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
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Applications in ecology
Recombinant Bacteria- bacteria which can be engineered to “eat” oil spills.
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Some Important Tools of Genetic Engineering
1) EnzymesRestriction Endonucleases (REs):DNA ligaseDNA PolymerasesReverse transcriptases
2)VectorsPlasmidBacteriophage, Cosmid Yeast
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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 dsDNA19
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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 bacteriumreplicate independent of the bacterial DNA.
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Bacteriophage
is a virus that can infect bacteria
Cosmidplasmid + 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) recognizes sequence
5’ GAATTC 3’.
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Action of EcoRI
Cuts both strand of the DNA
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Action of RE
Plasmid Plasmid with a cut
RE
DNA RE DNA fragment
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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). 27
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Applications of REs in Genetic Engineering1) 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). 28
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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.31
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 36
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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).
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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,
tPa, antihemophilic factor, interferon, etc.). Disease prevention
(e.g. vaccines, such as hepatitis B antigen)
Diagnostic tests
(e.g. monoclonal antibodies).40
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Human insulin is produced using Recombinant DNA Technique :
Recombinant Human Growth Hormone
Recombinant insulin (Humulin)
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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 PCRUseful : 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.
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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)51
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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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“To be or not to be, That is the question.”
- The tragedie of Hamlet, Prince of DenmarkeWilliam Shakespeare
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