Applications of Biotechnology in Medicine | Essay | Biotechnology

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Applications of Biotechnology in Medicine

Essay Contents:

1. Essay on Edible Vaccines
2. Essay on the Primary Structure of Insulin
3. Essay on Gene Therapy
4. Essay on Molecular Diagnosis

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Essay # 1. Edible Vaccines:

Vaccines of common use are usually produced by cell cultures or animals. Such vaccines contain weakened or inactivated pathogens. Crop plants can bear cheaper bioreactors to produce antigens which can be utilised as edible vaccines.

Even tissues of these transgenic plants can be eaten raw.

Advantages of such plants are:

(a) No storage problem.

(b) Easy delivery system by feeding.

(c) Cheap alternative as compared to recombinant vaccines produced by bacterial fermentation.

Antigens from several pathogens have been expressed in plants. In banana and tomato, such antigenic proteins have been expressed. Transgeruc banana and tomato can be consumed by patients suffering from cholera and hepatitis B. Foot and mouth disease of animals can be cured by feeding them transgenic sugar beet. In near future, these vaccines can be used as conventional vaccines. In such cases gene encoding antigen is isolated from pathogen and then transferred and expressed.

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Essay # 2. Primary Structure of Insulin:

The first protein whose amino acid sequence forming its primary structure was determined, was insulin. This protein is formed of two polypeptide chains—A- and B-chain, interlinked covalently by two disulfide bonds (Fig. 12.4). A-chain is formed of 21 amino acid residue, while B-chain is formed of 30 amino acid residue.

The A-chain had an N-terminal Glycine (GLY) and a C-terminal Asparagine (Asn), while the B-chain has an N-terminal Phenylalanine (Phe) and a C-terminal Alanine (Ala). Two disulfide bonds (-S-S- ) present between two chains lie between cysteine amino acids located at 7th and 20th position of A-chain and 7th and 19th position of B-chain, A third disulfide bond also occurs in the A-chain between cysteine (Cys) amino acids at 6th and 11th position (Fig. 12.4).

Both chains of insulin are biosynthesized as a single polypeptide chain, proinsulin (Fig. 12.5) in which A and B- chains are interlinked by a connecting polypeptide of 33 amino acids (Fig 12.4 and 12.5).

Its synthesis is controlled by the gene located on the short arm of chromosome 11. It then undergoes proteolytic processing forming insulin.

Primary structure of ribonuclease was determined by Hirs, Moore and Stein (1960). It consists of a single polypeptide chain of 124 amino acid residues with Lysine (Lys) at N-Terminus and valine (Val) at C-terminus. Eighty cysteine residue are joined by 4 covalent disulfide linkages.

Genetically Engineered Insulin:

Insulin is a proteinaceous hormone secreted by β-cells of Islets of Langerhans.

Sharpy-Shafer suggested that diabetes occurs due to failure of some islands of pancreas to secrete insulin.

Earlier insulin required for diabetes was extracted from pancreas of slaughtered cattle and pigs. The process was quite tiresome and difficult and yields of insulin would be low. In some patients it developed allergy or other side effects to foreign protein. In 1983 Eli Lilly an American Company prepared two DNA sequences for A and B chains of human insulin and introduced them m plasmids of E. coli. This led to production of insulin chains.

Eli Lilly has started selling humulin since 5th July 1983. Eli Lilly and Ranbaxy launched a new insulin project namely humalog (an analog of 5, 6 human insulin), which is more expensive than human insulin products. Humalog is absorbed within 10-15 minutes as against 30-60 minutes of other insulin products.

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Essay # 3. Gene Therapy:

Gene therapy is humans, is to replace ‘a faulty gene’ by a normal healthy functional gene. Gene therapy is a collection of methods which permits the correction of a gene defect which has been diagnosed in a child/embryo. In gene therapy normal genes are inserted into individual or embryo to take over the function and compensate for non-functional gene.

Under gene therapy, hereditary diseases like sickle cell anaemia to killer diseases like SCID (Severe combined immuno deficiency). SCID occurs due to defect in the gene for enzyme adenosine deaminase (ADA). Such patients bear non-functional T-lymphocytes.

As a result, they fail to mount immune responses against attacking pathogens. The first clinical gene therapy was given to a 4 year girl in 1990 with ADA deficiency. The ideal approach for SCID treatment is to provide functional ADA which breaks down toxins. The defect is caused due to deletion of gene for ADA synthesis.

It is cured in children by two methods:

(i) Bone marrow transplantation.

(ii) Enzyme replacement therapy.

However by applying above methods, disease is not completely cured.

Lymphocytes, a kind of white blood cells are extracted from bone marrow of a SCID sufferer. A good copy of human gene encoding this enzyme is introduced into such cells. This is done by gene therapy in which lymphocytes from blood are grown in culture outside the body. A functional ADA cDNA (using a retroviral vector) is introduced into such cells. They are reinjected into SCID sufferers.

Usually patients need regular infusion of genetically engineered lymphocytes. Because such cells are not immortal. But if a better gene is introduced into bone marrow cells at an early embryonic stage, a permanent cure can be achieved.

Clinical trials of diseases that are being considered for using somatic gene therapy:

1. Colon cancer

2. Cystic fibrosis

3. Acute myeloid leukaemia

4. AIDS

5. SCID

6. Breast cancer

7. Atherosclerosis

8. Haemophilia

9. Hypercholestromia

10. Leukaemia

11. Liver cancer

12. Lung cancer

13. Osteoporesis

14. Sickle cell anaemia 

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Essay # 4. Molecular Diagnosis:

For any disease, an early diagnosis by knowing symptoms, pathophysiology etc., is required. Recombinant DNA molecules and some techniques like Polymerase Chain Reactions have proved very rewarding in this regard. PCR technique has already been discussed. In ELISA (Enzyme linked immunosorbant assay) cloned genes are expressed to produce recombinant proteins help in developing sensitive diagnostic techniques.

Enzyme-Linked Immunosorbent Assay (ELISA):

Elisa technique is widely used for the detection of viruses, fungi, bacteria, mycoplasmas-like organisms, etc. It is a very sensitive and specific test, and needs very small amount of reagents. It is used to detect the infectious diseases like tuberculosis, AIDS, etc.

It was first developed by two groups of workers—Engvall and Perlamann, and Weeman and Schuurs in 1971. Vollar et. al (1976) and Clark and Adams (1977) used FT ISA for the detection of virus infection. In India, Usha M. Joshi of Mumbai also used it for detection of virus infection. More several modifications of original ELISA are known.

Principle and General Procedure:

ELISA is based on the ability of low molecular weight antibodies to couple with enzymes, to produce enzymatically active immunological conjugates. This allows the detection of immune reaction with histochemical staining techniques because the antibody component is involved in immune reaction and the conjugated enzyme can be used for staining reaction using appropriate substrate. It can be used to detect antigen as well as antibodies in the serum of patient.

In ELISA technique, a polystryrene or polyvinyl chloride microtitre plate is used, which has wells to provide a solid phase for immune reaction.

It involves the following steps (for the detection of specific antigens):

(a) ELISA – wells of polystyrene or polyvinyl chloride microtitres are coated with antibodies.

(b) Patient’s serum is added to the wells and incubated at 37°C for a specific period of time. Antigen- antibody complexes are formed at the bottom of wells. Excess of serum is washed by repeated washing of wells.

(c) Now enzyme-linked antigen to be detected is added to the wells and incubated at 37°C for a specific period. The enzyme being labelled gets attached to Antigen- antibody complex. Excess of enzyme is again washed away by repeated washings.

(d) Now substrate is added to the wells and incubated at 37°C for a specific period of time. Enzyme reacts with the substrate to form particular colour complex.

(e) Reaction is stopped with a provided solution of sulphuric acid.

(f) Reading is taken with an ELISA reader (Fig. 12.8).

(g) In this way, a number of readings are taken and by comparing them with a set of standard reading, inference can be concluded.

e.g. for Tuberculosis:

If ELISA reader value is > 350 sero unit/ml = + ve

If < 200 sero unit/ml = – ve

If between 200 – 350 sero unit/ml = Equivocal.

ELDNA Probes:

DNA probe is a small DNA segment (20-50 bases long) that recognizes complementary sequence in DNA molecule and thus, allow identification and isolation of specific DNA sequence from an organism.

Uses of DNA Probes:

1. They are used in genetic engineering. For identification of recombinant clone carrying desired DNA insert.

2. They are frequently used for variety of purposes including diagnosis of infectious diseases, identification of food contaminants, variety of microbial tests, forensic test.

3. Probes can also be used to identify strains of an organism e.g., varieties of crop species.

4. Development of RFLP maps.

5. For preparation of genome maps in eukaryotes.

6. It can be used to detect and locate its complementary sequence in hybridization experiments.

Preparation of a Probe:

From genomic DNA (DNA extracted from cell nuclei), a DNA sequence (probe) is isolated using following steps:

(i) Extract DNA from cells.

(ii) Digest this DNA with restriction enzymes like Eco RI or Hind III. This will cut DNA on specific positions.

(iii) Run the digested DNA on agarose gel for electrophoresis. This will separate out the segments of different sizes.

(iv) Isolate DNA of particular segment size from a specific band identified with Southern plots by hybridization with specific labelled m RNA or c DNA molecules.

(v) Clone this DNA in a vector.

(vi) Permit chimeric vector to infect bacteria for rapid multiplication. From this transformed bacteria, chimeric vector can be used directly as probe.

(vii) For detection of homologous sequences after hybridization with the probe is like finding a needle in the haystack.

For success of DNA probe assay either of two methods are employed:

(a) As probes transmit no signal of their own they have to labelled with radioactive isotopes.

(b) Signal molecules are used which may include flourescent antibodies and enzymes that produce colour changes in dyes and chemiluminescent catalysts.

In agarose gel electrophoresis experiment, it is to be stained with gel with a compound called ethidium bromide to make DNA visible. But in this method, small segment of DNA cannot be easily identified with this staining technique. For this, DNA probe is labelled by inserting nucleotides having radioactive isotope of phosphorus or sulphur or a florescent molecule.

Application of Molecular Diagnosis:

1. Very low concentration of bacteria or virus (pathogen) at the time, when symptoms of disease are not visible can be detected by amplification of their nucleic acids by Polymerase Chain Reaction (PCR).

2. PCR technique is being widely used to detect HIV in suspected AIDS patients.

3. PCR technique is used to detect mutations in suspected cancer patients.

4. Cloned genes are used as probes to detect the presence of its complementary DNA strands.

5. ELISA technique is widely used to detect viruses, fungi, bacteria, mycoplasma like organisms.

6. ELISA test is also used to diagnose diseases like tuberculosis, AIDS etc.