1 r dna & its pharmaceutical applications

Overview of recombinant DNA technology

Bacterial cell

Bacterialchromosome

Plasmid

Gene of interest

DNA containinggene of interest

Isolate plasmid.

Enzymatically cleaveDNA into fragments.

Isolate fragmentwith the gene ofinterest.

Insert gene into plasmid.

Insert plasmid and gene intobacterium.

Culture bacteria.

Harvest copies ofgene to insert intoplants or animals

Harvest proteinscoded by gene

Eliminateundesirablephenotypictraits

Produce vaccines,antibiotics,hormones, orenzymes

Createbeneficialcombinationof traits

Applications of Recombinant DNA Technology

• Pharmaceutical and Therapeutic Applications1. Protein synthesis

• Creation of synthetic peptides for cloning• E.g. Human Insulin, Human Growth Hormone

© 2012 Pearson Education Inc.

Ligase

E. Coli

E. Coli

2. Vaccines• Production of safer vaccines• Subunit vaccines• Genes of pathogens introduced into common fruits and

vegetables• Injecting humans with plasmid carrying gene from

pathogen– Humans synthesize pathogen’s proteins


Recombinant vaccines can be broadly grouped into two kinds:

(i) Recombinant protein vaccines: This is based on production of recombinant DNA which is expressed to release the specific protein used in vaccine preparatione.g. Hepatitis B, cholera, diphtheria, tetanus vaccines

(ii) DNA vaccines: Here the gene encoding for immunogenic protein is isolated and used to produce recombinant DNA which acts as vaccine to be injected into the individual.e.g. Live vaccinia virus vaccine against small pox

-Vaccines

Production of recombinant vaccines involves the following steps:

(i) First and foremost, it is important that the protein which is crucial to the growth and development of the causative organism be identified.

(ii) The corresponding gene is then isolated applying various techniques. Further to this, an extensive study of the gene explains the gene expression pattern involved in the production of corresponding protein.

(iii) This gene is then integrated into a suitable expression vector to produce a recombinant DNA.

(iv) This rDNA is used as vaccines or is introduce into another host organism to produce immunogenic proteins which acts as vaccines.

Advantages:(i) Since it does not involve actual pathogen, recombinant vaccines is considered to be safe than the conventional vaccines.(ii) It induces both humoral and cellular immune response resulting in effective vaccination.

Risks involved:(i) High cost of production.(ii) Have to be stored at low temperature since heat destabilizes protein. Hence storage and transportation is tedious.(iii) Individuals with immunodeficiency may elicit poor immune response.

-Vaccines

3. Genetic screening/ testing• DNA microarrays used to screen individuals for inherited

disease caused by mutationsE.g. Huntington's disease

• Can also identify pathogen’s DNA in blood or tissues

4. DNA fingerprinting• Identifying individuals or organisms by their unique DNA

sequence


DNA fingerprinting

5. Gene therapy• Uses genes to treat or prevent disease.• Missing or defective genes replaced with normal copies• A number of viruses have been used for human gene

therapy, including retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia, pox virus, and adeno-associated virus

• Trials are going on for treatment of cancer and other genetic disorders using gene therapy


Gene therapy

6. Medical diagnosis• Amniotic fluid or Chorionic villi (tiny, finger-shaped

growths found in the placenta) sampling is done during pregnancy most commonly to check for disorders such as Down’s syndrome. Also for diagnosis of other metabolic or hematological disorders.


7. Xenotransplants– Xenotransplantation, is the transplantation of animal

cells, tissues or organs into human body. Such cells, tissues or organs are called xenografts or xenotransplants– A worldwide shortage of organs for clinical implantation causes

about 20–35% of patients who need replacement organs to die on the waiting list. Certain procedures, some of which are being investigated in early clinical trials, aim to use cells or tissues from other species to treat life-threatening and debilitating illnesses such as cancer, diabetes, liver failure and Parkinson's disease.


• Environmental Studies– Most microorganisms have never been grown in a

laboratory– Scientists know them only by their DNA fingerprints

• Allowed identification of over 500 species of bacteria from human mouths

• Determined that methane-producing archaea are a problem in rice agriculture



• Agricultural Applications– Production of transgenic organisms

• Recombinant plants and animals altered by addition of genes from other organisms


• Agricultural Applications– Herbicide tolerance

• Gene from Salmonella conveys resistance to glyphosate (Roundup™)

– Farmers can kill weeds without killing crops

– Salt tolerance• Scientists have inserted gene for salt tolerance into

tomato and canola plants• Transgenic plants survive, produce fruit, and remove

salt from soil


• Agricultural Applications– Freeze resistance

• Crops sprayed with genetically modified bacteria can tolerate mild freezes

– Pest resistance• Bt toxin

– Naturally occurring toxin harmful only to insects – Organic farmers used to reduce insect damage to crops

• Gene for Bt toxin inserted into various crop plants• Genes for Phytophthora resistance inserted into potato

crops


• Agricultural Applications– Improvements in nutritional value and yield

• Tomatoes allowed to ripen on vine and shelf life increased

– Gene for enzyme that breaks down pectin suppressed• BGH allows cattle to gain weight more rapidly

– Produce meat with lower fat content and produce 10% more milk

• Gene for β-carotene (vitamin A precursor) inserted into rice

• Scientists considering transplanting genes coding for entire metabolic pathways


Application of recombinant dna technolgy

• Recombinant bacteria in Industry• Recombinant bacteria in Medicine• Recombinant bacteria in Agriculture• Production of Transgenic animal• Production of Transgenic plants• Bioprocessing (A bioprocess is specific process that uses

complete living cells or their components (e.g., bacteria, enzymes, chloroplasts) to obtain desired products.)

1 r dna & its pharmaceutical applications

Health & Medicine