Gene cloning

RISHABH GARG (BE ECE) BITS GOA
GENE
RISHABH GARG
First Degree Program (ECE)
Birla Institute of Technology & Science,
K.K. Birla Campus Goa 403 726 IN
CLONING

Recombinant DNA
• Recombinant DNA is the formation of a
novel DNA sequence by the combination
of two DNA fragments
• These are taken from two different
organisms

• The method by which DNA of the donor
organism (target DNA) is cut into
fragments with the help of Restriction
Enzymes and insert one of these
fragments into the DNA of the host.
r - DNA Technology

• Hamilton Smiths (1970) at Johns
Hopkins Medical School isolated the first
RE that cut DNA at a very specific
nucleotide sequence.
• Stanley Cohen & Herbert Boyer (1972)
created Recombinant DNA.
Genesis

Target DNA (Deoxyribonucleic acid)
 DNA is the hereditary
material passed on
from generation to
generation.
 DNA is a long double-
stranded chain of
nucleotides
 DNA is made up of four
nucleotides: A, C, G & T.
Requisites for r-DNA

 A always pairs with T
 C always pairs with G
 Two complementary
DNA strands will
separate when heated,
and will spontaneously
pair together again
when cooled.
Target DNA (Deoxyribonucleic acid)

Tetr
Ampr
Ori
pBR322
4361bp Ori
pUC18
Ampr
MCS
LacZ
• Vector is a small piece of DNA into which
a foreign DNA fragment can be inserted
Cloning Vector

• Plasmids are naturally occurring extra-
chromosomal DNA molecules.
• Plasmids are circular, double-stranded DNA.
• Plasmids can be cleaved by restriction
enzymes, leaving sticky or blunt ends.
• Artificial plasmids can be constructed by
linking new DNA fragments to the sticky ends
of plasmid.
Plasmid as a Cloning Vector

• Restriction Endonuclease is an Enzyme that
cuts DNA (both single as well as double
stranded) at specific nucleotide sequences
known as restriction sites
Restriction Enzymes

Recognition site
Cuts usually occurs at a
palindromic sequence
SmaI: produces blunt ends
5´ CCCGGG 3´
3´ GGGCCC 5´
EcoRI: produces sticky ends
5´ GAATTC 3´
3´ CTTAAG 5´
DON'T NOD
DOGMA: I AM GOD
NEVER ODD OR EVEN
TOO BAD – I HID A BOOT
RATS LIVE ON NO EVIL STAR
NO TRACE; NOT ONE CARTON
WAS IT ELIOT'S TOILET I SAW
MURDER FOR A JAR OF RED RUM
SOME MEN INTERPRET NINE MEMOS
CAMPUS MOTTO: BOTTOMS UP, MAC
GO DELIVER A DARE, VILE DOG
MADAM, IN EDEN I'M ADAM
OOZY RAT IN A SANITARY ZOO
AH, SATAN SEES NATASHA
LISA BONET ATE NO BASIL
DO GEESE SEE GOD
GOD SAW I WAS DOG
DENNIS SINNED

• Catalyze joining or recombine the two
fragments of DNA - a process known as
ligation.
DNA Ligase Enzyme

Re-joined by Ligase
Enzyme
Steps involved in r-DNA
• Both target DNA and the Vector are cut with a RE
• The Restriction Enzyme leaves sticky ends
• The sticky ends bind both the DNA together
• Ligase seals the DNA

Steps involved in r-DNA
• DNA is taken up by the Host Cells
• As the Host Cells grow, DNA also grow simultaneously.

An overview of r-DNA

r-DNA : A Video

• Three types of libraries can be developed :
• DNA Or Genomic Libraries
• cDNA Libraries from mRNA
• Expression Libraries to express foreign proteins
Genetic Libraries

 A Genomic Library contains
all the Chromosomal DNA
of a cell
 DNA is purified and
fragmented into pieces
carrying thousands of
bases.
 The size of fragments
depends on the capacity of
Vector to accommodate
and propagate the DNA.
DNA Library

 A c-DNA Library contains
all the sequences of mRNA
found in a cell
 The single stranded mRNA
molecule is converted into
double stranded DNA
through the action of
reverse transcriptase.
 They lack transcriptional
control and intronic
sequences found in
genomic clones.
c-DNA Library

 Again a c-DNA Library in a
special form of vector that
permits transcription of
the incorporated c-DNA.
 Eukaryotic proteins are
formed in bacterial host
cells that are normally not
present in prokaryotes.
 Proteins can be made and
purified more easily in
bacterial cells.
Expression Library

Gene Expression

Laboratory Applications
• Structure & Function of a gene in isolation and in
varied juxtapositions.
Commercial & Therapeutic Applications
• Therapeutic Cloning
• Reproductive Cloning
Applications

Therapeutic Cloning
• Therapeutic Cloning is performed to harvest embryonic
stem cells. These cells are found inside the developing
embryo and they can be used to develop tissues & organs.

• A cell is removed from the patient’s body. Its nucleus is
taken out and inserted into an enucleated egg cell.
• Cell division is triggered either chemically or through
electric shock. The resulting embryonic stem cells are
then removed and used to treat the patient.
Therapeutic Cloning

Therapeutic Cloning
 Therapeutic Cloning
can help in tissue or
organ transplantation.
 The Embryonic Stem
Cells can generate a
genetically 100%
compatible tissue or
organ.
 The donation of
tissues and organs not
required in such
cases.

Gene Knockout

Reproductive Cloning
• Reproductive Cloning refers to create an exactly identical
or vegetative copy of an organism.
• It is performed using a technique known as SCNT -
Somatic Cell Nuclear Transfer

Reproductive Cloning
• The ovum of the animal to be cloned is enucleated.
• Then a cell is taken from the same organism and its
nucleus is removed.
• This nucleus is then transferred into the enucleated egg
cell.
Sir Ian Wilmut
who cloned
Dolly - a sheep
1996 - 2003

• Even after revelation of entire
human genome, our
understanding about human
genome is quite trivial
• Only 1.5% of the entire genome
contains genes. The rest,
overlooked so far as ‘junk DNA’,
may have also a bearing on the
expression of genes.
• There is no such process like
meiosis (crossing-over) which
occurs during normal sex-cycle.
Human Clones

Cloning : A Video

Thank You
for the active listening
“Genetic cloning is a powerful tool and the
maturity, with which we tackle it, would
decide the fate of human race.”

Gene cloning

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