Recombinant DNA Technology

PRESENTED BY:D.PRIYANKAM-PHARMDEPARTMENT 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 DNA

that codes for a type of protein that has a function in the organism.

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

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

Gene cloningIt 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.