01 Recombinant Dna Technology

See discussions, stats, and author profiles for this publication at: https://www.researchgate.
net/publication/325269753
Recombinant DNA Technology
Presentation · October 2013
DOI: 10.13140/RG.2.2.21423.64167
CITATION READS
1 6,359
1 author:
H.K. Garg
Institute for Excellence in Higher Education, Bhopal
91 PUBLICATIONS 225 CITATIONS
SEE PROFILE
Some of the authors of this publication are also working on these related projects:
Growth Inhibition of Mycobacterium tuberculosis complex by Andrographolide and its effect on A-549 & L-132 Cell lines. View project
UGC Sponsored Research Project on Genomic Transforms in Birds View project
All content following this page was uploaded by H.K. Garg on 21 May 2018.
The user has requested enhancement of the downloaded file.
Professor
Cell & Molecular Biology and Genetics
Sarojini Naidu Government Girls

Post Graduate Autonomous College
Bhopal - 462016
Cell Phone - 94244 17792

Email - drhkgarg@gmail.com
Professor H.K. Garg 17 Oct 2013
• 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.
• 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.
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.
Target DNA (Deoxyribonucleic acid)
§ 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.
Coning Vector
• Vector is a small piece of DNA into which
a foreign DNA fragment can be inserted
LacZ
Ampr MCS
Ori
pBR322 pUC18
4361bp Ori
Ampr
Tetr
Plasmid as a Coning 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.
Restriction Enzyme
• Restriction Endonuclease is an Enzyme that
cuts DNA (both single as well as double
stranded) at specific nucleotide sequences
known as restriction sites
Recognition site DON'T NOD

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

• Catalyze joining or recombine the two fragments
of DNA - a process known as ligation.
• 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
Re-joined by Ligase
Enzyme
• DNA is taken up by the Host Cells

• As the Host Cells grow, DNA also grow simultaneously.
• Three types of libraries can be developed :

• DNA Or Genomic Libraries
• cDNA Libraries from mRNA
• Expression Libraries to express foreign proteins
• 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.
• A cDNA 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.
• Again a cDNA Library in a

special form of vector that
permits transcription of the
incorporated cDNA.
• 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.
Laboratory Applications
• Structure & Function of a gene in isolation
and in varied juxtapositions.
Commercial & Therapeutic Applications
• Therapeutic Cloning
• Reproductive 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
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.
• 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
• 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 a
sheep - DOLLY
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.
View publication stats
“Genetic cloning is a powerful tool and

the maturity, with which we tackle it,
would decide the fate of human race.”
Thank You
for the active listening

01 Recombinant Dna Technology

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

01 Recombinant Dna Technology

Uploaded by

Copyright:

Available Formats

See discussions, stats, and author profiles for this publication at: https://www.researchgate.

Sarojini Naidu Government Girls

Cell Phone - 94244 17792

• Recombinant DNA is the formation of a

• The method by which DNA of the donor

• Hamilton Smiths (1970) at

Target DNA (Deoxyribonucleic acid)

§ DNA is the hereditary

Target DNA (Deoxyribonucleic acid)

§ A always pairs with T

Plasmid as a Coning Vector

Recognition site DON'T NOD

DNA Ligase Enzyme

• Both target DNA and the Vector are cut with a RE

• DNA is taken up by the Host Cells

• Three types of libraries can be developed :

• A Genomic Library contains

• A cDNA Library contains all

• Again a cDNA Library in a

• Therapeutic Cloning is performed to harvest embryonic stem

• Reproductive Cloning refers to create an exactly identical or

• The ovum of the animal to be cloned is enucleated.

Sir Ian Wilmut

• Even after revelation of entire

• Only 1.5% of the entire

• There is no such process like

“Genetic cloning is a powerful tool and

Professor H.K. Garg 17 Oct 2013

You might also like