Recombinant DNA technology and its applications

By

B.Ananthagowri

Recombinant DNA technology

• One of the recent advances in biotechnology, developed by two scientists named Boyer and Cohen in 1973.

• rDNA is a hybrid DNA produced by joining DNA fragments of two different organisms.

• The organism containing rDNA is called recombinant or genetically modified organism.

Various steps in the preparation of rDNA

• 1.preparation of desired gene – By three ways

• a. Desired gene is isolated from the total genomic DNA of an organism and cut in to pieces by restriction endonucleases.

• b. Desired gene is prepared from mRNA by reverse transcription.

• Reverse transcriptase

• mRNA cDNA

• c. synthesized by DNA synthesizer

2. Isolation of cloning vector

• Vector is a DNA which is used to transfer the desired DNA into a host cell.

• Plasmid – circular, double stranded, extra chromosomal DNA of bacteria.

• Plasmids are 1Kbp – 20Kbp. Bacteria have more than one copy of each plasmid (copy number).

• Plasmid number is increased by adding chloramphenical.• Some plasmids are isolated from bacteria and used directly

such plasmids are natural plasmids• Col E1 – E.coli• YEP and YIP – Yeast• Natural plasmids are large in size, have no genetic markers

and confer pathogenicity to the host.• Based plasmids or constructed plasmids have much use in

gene transfer.• PBR322, RSF1010, PSC101, PUC8.•

PBR322

• The size of this plasmid is 4.4Kbp. It has two gene markers

• Tetracyclin resistant gene and Amphicillin resistant gene.

• Copy number is 15, increased up to 3000.

• Bacterial cell can uptake 15Kbp size. So it carry large DNA segment of 5-10Kbp.

• Human GH gene introduced in to E.coli by this vector.

• Regulation and expression is good

DNA of bacteriophages

• Like plasmid, wild type of phage DNA carry small segment of foreign DNA.

• A non – essential sequence is deleted from the wild DNA and used as cloning vehicle.

cosmids

• Artificial plasmid. It is a combination of bacterial plasmid DNA and cos portion of bacteriophage DNA.

• It does not code for phage proteins and host cell lysis.

• It has an origin of replication from plasmid and selectable marker genes

3. Construction of rDNA

• Desired gene is inserted into the vector DNA by using the enzyme ligase.

• ligase

• Desired gene + vector DNA rDNA

• Splicing

Bio-pharmaceutical products of rDNA technology

• Hormones

• Insulin - Diabetes mellitus

• GH - Dwarfism

• calcitonin – treating osteoporosis

• Relaxin – facilitate child birth

• Interferones – antiviral protein(cold, influenza and hepatitis)

• Erythropoietin – treating anaemia

• Clotting factors VIII – treating haemophilia

• TPA, steptokinase and urokinase – dissolve blood clot, treating heart attack

• TPA – Tissue plasminogen activator

• Vaccines – Hepatitis B

About insulin

• It is a peptide hormone. Lowers blood glucose level.

• It is a dimer of A & B chain.

• A chain is made up of 21 amino acids.

• B chain is made up of 30 amino acids. Two chains are linked by disulphide bond.

• Insulin gene is present in the short arm of 11th chromosome. Gene length is 153 nitrogenous base pair.

Production of Genetic Engineered Insulin (Humulin)1. Human insulin producing gene is isolated from pancreatic cells.2. A plasmid DNA is extracted from a bacterium and cut with restriction enzyme, forming plasmid vector. 3. Insert human insulin-producing gene into the bacterial plasmid vector to form the recombinant DNA . 4. Introduce this recombinant DNA into a bacterial cell to form the recombinant bacterium.5. The recombinant bacteria multiply in a fermentation tank and produce A and B chains of human insulin.6.A &b chains are extracted, purified and mixed together and connected with each other by disulphide bond.7. Nowadays insulin is produced in yeast cells instead of E.coli because it secretes insulin molecule with perfect 3 dimensional structures.

Human growth hormone

• It is a protein hormone secreted by the anterior lobe of the pituitary.

• It is a single chain protein , made up of 191 amino acids.

• In children its deficiency leads to dwarfism

Production of Human Growth hormone1. A gene that produces growth hormone in humans is isolated or produced by reverse transcription.2. The growth hormone production gene is inserted into the DNA of E.Coli bacteria. 3. The bacteria recognize the inserted DNA as its own DNA and begin to produce human growth hormone. 4. The bacteria multiply and produce the growth hormone in a culturing media.5. The growth hormone is extracted and purified and is then ready to be injected into children with GHI.

Erythropoietin(EPO)

• It is a glycoprotein produced in kidney .

• It acts on bone marrow to increase the production of RBC & WBC. Stimuli such as bleeding or moving to high altitudes triggers the release of EPO.

• Used in the treatment of anaemia

• The EPO is produced by rDNA technology and widely used in AIDS for development of immunity.

• It is abused in sports to improve endurance.

Recombinant hepatitis B vaccine

Old method• Hepatitis B virus attacks the cells

of the liver, causing chronic liver failure, liver cancer and death. It can be prevented by vaccination.

• For many years the vaccine was produced by growing viruses in animals. The harvested viruses were inactivated by chemical treatment.

• In the above said method there is a risk of infection during production.

New method

• The gene coding for hepatitis

B surface antigen is isolated

and cloned in to a vector.

• The cloned gene transferred to

the yeast expression system.

• The recombinant protein

product is obtained, purified

and used for vaccination

Transgenic plants

• Insect resistant plant.

• Drought, cold and salinity resistant plant .

• Improvement of nutritional quality.

• Plant that produce polythene.

• Fluorescent plant.

Bt Maize

• Bacillus thuringiensisproduces a bacterial toxin (Delta-endotoxin)

• The bacterium has been used as an insecticide since 1938

• It stops the insect from feeding by attacking the insect gut lining

Bacillus thuringiensis

© 2010 Paul Billiet ODWS

Cry gene

• The toxin is produced by the Cry gene

• Found on plasmids in the bacterium

• The gene is added to the genomes of crop plants using a bacterium that forms root nodules in plants (Agrobacterium tumifaciens)

• Bt crops produced from 1996 onwards: maize, potato, cotton, soybean

© 2010 Paul Billiet ODWS

I

BT Maize

Golden rice with provitamin-A

• Developed by Dr. Ingopotryces and Peter Beyer.

• Two genes from the plant daffodil and one gene from bacteria(Erviniauredovora) involved in the production of beta carotene

Improved storage protein and sweet protein• Amaranthus grain have seed storage protein AMA-I which is rich in all

essential amino acids.

• cDNA of AMA-I gene is introduced in to potato calli.

• Transgenic potato contain higher level of essential amino acids.

• Monellin is a sweet protein present in the fruits of African plant

Dioscorephyllumcumminsii. It ia 100000 times sweeter than sucrose.

• The gene coding for monellin is isolated and introduced in to lettuce plant.

Transgenic lettuce produces monellin.

• The same gene is introduced in to tomato through Ti plasmid.

• Monellin content is less in immature fruits but very high in ripe fruits.

• Thaumatin is a sweet protein present in the fruits of Thaumatococcus

danielli– endemic plant of Western Africa.

• cDNA of thaumatin was made from mRNA and cloned in E.coli and Yeast

• GMO produce thaumatin in large quantity and it is sold in the name of

talin.

• Talin is a boon to diabetcs who could not taste sugars.

Stress tolerant plants

• Cold tolerance

• Arabidopsis is a plant contain acyl glycerol phosphateAGP) to

resist chillness.

• Glycerol phosphate acyl transferace

• Glycerol diphosphate AGP

• The GPAT gene is introduces in to Tobacco through Ti

plasmid. Transgenic tobacco resist chilling as low as 10ºc.

• Drought resistant plants.

• Genes coding for sorbitol and mannitol dehydrogenase are

isolated from E.coli and introduced in to drought sensitive

plants.

• Transgenic plant produce sorbitol and mannitol and hence

resist drought and heat.

Transgenic plants as bioreactors

• Bio reactors are culture vessels used for large scale production of some valuable products.

• Some transgenic plants are used for the large scale production of vaccines, interferons, biodegradable plastics etc.

• Gene for protein coat of hepatitis virus was isolated and introduced in to banana. The banana is rich in the antigenic protein.

• Human interferon gene is introduced in to tobacco.

• Polyhydroxybutyrate is a plastic like natural polymer produced in bacteria.

• Biosynthesis is carried out by three enzymes.

• 1.3- ketothiolase present in all plants

• 2.acetyl co-A reductase – Alcaligenes eutropus

• 3.PHB synthase - ”

• Two genes isolated from bacteria is introduced in to Arabidopsis thaliana.

• The transgenic plant produce 0.2 to 0.5 µg PHB/gram in their cells.

A light-producing plant called Starlight Avatar that glows like a firefly has been created by U.S. scientists. It is pictured in daylight (left) when it looks like a regular pot plant, but it can be seen to emit light in darkness