Gene Cloning ...... Rishabh Garg

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´

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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

https://www.youtube.com/watch?v=MIfDx417SDs

https://www.youtube.com/watch?v=MIfDx417SDs

• 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

https://www.youtube.com/watch?v=-kHOEtaWntA

https://www.youtube.com/watch?v=-kHOEtaWntA

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 ...... Rishabh Garg

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