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

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YES...!!!!!!!!!!!!

• 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.

hhhgenetic

Genetic EngineeringOR

Recombinant DNA Technology

Dr. Gangadhar ChatterjeeMBBS;MD

Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India

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

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.

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.

.

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 dsDNA19

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?

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

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

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.

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

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)

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

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

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

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

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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