Recombinant DNA

Technology

PRESENTED BY:

D.PRIYANKA

M-PHARM

DEPARTMENT OF PHARMACEUTICS

UNDER GUIDENCE OF:

Mrs.YASMIN BEGUM

ASSOCIATE PROFFESSOR(Ph.D)

MALLA REDDY COLLEGE OF PHARMACY

CONTENTS:

DefinitionRECOMBINANT dna TECHNOLOGYRESTRICTION ENZYMES AND PLASMIDSDEFINITION OF GENE GENE CLONINGBASIC STEPS IN GENE CLONINGAPPLICATIONS OF rdna technologyConclusionreferences

What is DNA?DNA= Deoxyribu-Nucelic Acid

DNA is a very large molecule,

made up of smaller units called

nucleotides

Each nucleotide has three parts: a

sugar (ribose), a phosphate

molecule, and a nitrogenous base.

The nitrogenous base is the part of

the nucleotide that carries genetic

information

The bases found in DNA are four:

adenine, cytosine, guanine, and

thymine ( ATP, CTP, GTP, and

TTP)

Recombinant DNA Technology

Recombinant DNA technology

procedures by which DNA from

different species can be isolated, cut

and spliced together -- new

"recombinant " molecules are then

multiplied in quantity in populations of

rapidly dividing cells (e.g. bacteria,

yeast).

Recombinant DNA Technology

In the early 1970s it became possible to

isolate a specific piece of DNA out of the

millions of base pairs in a typical genome.

Recombinant DNA Technology

Recombinant DNA technology is based on a

number of important things:

Bacteria contain extra chromosomal

molecules of DNA called plasmids which

are circular.

Recombinant DNA Technology

Bacteria also produce enzymes called

restriction endonucleases that cut DNA

molecules at specific places into many smaller

fragments called restriction fragments.

There are many different kinds of restriction

endonucleases

Recombinant DNA Technology

Restriction Enzymes and plasmid

Sticky end and blunt end are the two

possible configurations resulting from the

breaking of double-stranded DNA

Recombinant DNA Technology

Restriction Enzymes and plasmid

When RES acts at the center of symmetry, two

complementary strands of DNA are of equal

length, hence forms the blunt end.

T C A G A T C A GA

A G T C T A G T CT

Recombinant DNA Technology

Restriction Enzymes and plasmid

Some RES breaks the DNA on either side of

center of symmetry with the liberation of

unequal fragments which are called as stick

ends/ cohesive ends.

G A A T T C G A A T T C

C T T A A G C T T A A G

Recombinant DNA Technology

Digestion of DNA by EcoRI to produce

cohesive ends.

Recombinant DNA Technology

Restriction Enzymes and plasmid

Restriction Enzymes are primarily found in bacteria and are given abbreviations based on genus and species of the bacteria.

One of the first restriction enzymes to be isolated was from EcoRI

EcoRI is so named because it was isolated from Escherichia coli strain called RY13.

What is gene?

• A gene is a stretch of DNAthat codes for a type of protein that has a function in the organism.

• It is a unit of heredity in a living organism.. All livingthings depend on genes

• Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring.

Gene cloning It can be defined as the isolation and

amplification of an individual gene

sequence by insertion of that individual

gene sequence into a bacterium where it

can be replicated

BASIC STEPS IN GENE CLONING

Step 1

A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a chimera or recombinant DNA (rDNA) molecule.

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Step 2

The vector acts as a vehicle that transports the gene

into a host cell, which is usually a bacterium

although other types of living cell can be used. This

process is called transformation.

Step 3

Within the host cell the vector multiplies producing

numerous identical copies not only of itself but also

of the gene that it carries.

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Step 4

When the host cell divides, copies of rDNA molecule are passed to the progeny and further vector replication takes place.

Step 5

After large no: of cell divisions a colony or clone of identical host cells is produced. Each cell in the clone contains one or more copies of the rDNA molecule

Step 6

Then, the host cells are then lysed and rDNA can be separated.

Recombinant DNA technology had made it possible to treat

different diseases by inserting new genes in place of

damaged and diseased genes in the human body.

Applications of rdna technology in medicine

Insulin is a hormone made up of protein. It is secreted in

the pancreas by some cells called as islet cells. If a

person has decreased amount of insulin in his body, he

will suffer from a disease called diabetes. Recombinant

DNA technology has allowed the scientists to develop

human insulin by using the bacteria as a host cell and it

is also available in the market. It is believed that the

drugs produced through microbes are safer.

Insulin:-

VACCINES:

Recombinant DNA technology enables the

scientists to develop vaccines by cloning the gene used

for protective antigen protein. Viral vaccines are most

commonly developed through this technology for

example, Herpes, Influenza, Hepatitis and Foot and

Mouth Diseases

Human Growth Hormones:-

In recent years, scientists have developed many growth

hormones using recombinant DNA technology. The

disease of dwarfism is treated with this hormone.

Infectious Diseases:-

Many diseases are diagnosed by conducting certain tests.

Recombinant DNA technology has allowed the development

of many tests which are being used to diagnose diseases like

TB and cancer.

In the diagnosis process, certain pathogens are isolated and

identified, and then diagnostic kits are produced when the

genome of the specific pathogen is known to kill it or block

its pathogenic activity.

PRODUCTION OF NOVEL PLANTS:

Rdna is used in distinguishing of novel agricultural plants

which are high yielding and pest resistant

Cloning of genes from wild pest resistant varieties has been

used.

Strain improvement for fermentation:

Rdna uses extensively for improvement of strains of

microbes.

REFERENCES:

FUNDAMENTALS OF MEDICAL

BIOTECHNOLOGY: Author: Aparna Raja

Gopalan,editors: irfan ali khan, page no:203-226

U.Sathyanarayana: biotechnology: page no: 530-542

pharmaceutical biotechnology: fundamentals and

applications: Author: s s kori. Page no:74-80.