Biotechnology And Its Applications

Bt cotton is a genetically modified plant which is resistant towards the pest called bollworm. It contains the Bt toxin gene from the bacterium, Bacillus thuringiensis (Bt). The Bt toxins kill a huge range of pesticides like larvae of moths and butterflies, bollworms, beetles, etc. But it is not resistant to all kinds of pesticides.

C-peptide of human insulin is a component used in the production of human insulin by recombinant DNA technology. The C-peptide is a short protein chain which is used to connect the A and B – peptide chains which are present in E.coli. After connecting the A and B peptide chains with disulphide bonds the C-peptide is removed during maturation of pro-insulin to insulin.

The Genetic Engineering Approval Committee (GEAC) is an organization set up by the Indian government which makes important decisions like the safety of the introduction of new genetically modified organisms (GMO) to the public and regulating GM research.

α -1 antitrypsin is a human protein which is used for the treatment of emphysema disease. It is a disease caused by the inflammation of alveoli present in the lungs. α -1 antitrypsindeficiency can lead to emphysema. It can be produced by transgenic organisms and used to treat emphysema.

A probe is an RNA or DNA the fragment which is used for labeling of DNA sequences for genomic mapping and sequencing. A probe is generally radiolabelled or fluorescent labeled and detected by several techniques.

Retroviruses are either single or double-stranded RNA viruses which infect their hosts and insert their gene copies in the host’s DNA and changes its genome which generally causes lysis of the host.

An example of a retrovirus is the HIV.

The site of production of the enzyme ADA (i.e., adenosine deaminase) in the lymphocytes which are formed in the lymphoid tissues present in the thymus and lymph nodes.

The word pro in protoxin indicates its inactive form.

Diseases caused by the pathogen causes change in the physiology of the host. E.g. swelling, lysis of cell etc.

• Protoxins on getting alkaline environment gets activated.

• It binds with the gut lining and creates pore in the gut.

• Golden rice is the nutritionally modified crop, rich in vitamin A and β-carotene.

• Gene for β-carotene is taken from daffodil and inserted into the rice plant.

• RNAi involves the transcription of sense and antisense sequence of DNA, which forms two complementary RNA strands which combines to form ds RNA.

• ds RNA are chopped of and there expression gets silenced.

In this condition enzyme ADAase is absent. These enzymes are needed for the maturation of the lymphocyteswhich ared employed in immune system for defence purpose.

Its deficiency results in SCID the genetic disorder.

Both the sense and antisense DNA undergoes transcription to form double stranded RNA, which silences the gene expression of nematode.

Green revolution began during the 1960s and 1965s to increase the production of food grains in India. It involved the use of High yielding variety (HYV) seeds and better practices and management of pesticides for pest control. Despite its success in the increase of food grains it had some of the following limitations:

• It wasn’t helpful for poor farmers as the HYV seeds and pesticides were costly. It led to farmer debts.

• The green revolution was only limited to food grain crops which majorly focused on wheat and rice only.

• The high yielding variety (HYV) required additional water and fertilizers for giving good crop yields.

These were the major limitations of the Green revolution.

GMO stands for genetically modified organism. The Genetically modified organisms are different from hybrids as:

• Hybrids are produced by cross breeding or cross pollinating two different animal or plant species, whereas the production of GMO involves insertion of a gene into a new organism so as to express a specific trait or character.

• GMO involves a more specialized set up compared to hybrids which are much more easily produced.

Diagnostics are the various methods or techniques which are involved in the diagnosis or detection of disease, For example, ELISA (i.e., Enzyme-Linked Immunosorbent Assay) is a diagnostic technique used for the detection of HIV, Hepatitis B, and Hepatitis C.

Therapeutics involves various agents which help in the treatment for a particular disease. For example, Ampicillin which is an antibiotic helps in the treatment of bacterial infections in humans is a therapeutic agent.

The full form of ELISA is Enzyme-Linked Immunosorbent Assay which is a diagnostic method which is used for the detection of the following diseases:

• HIV (i.e., Human Immunodeficiency Virus)

• Hepatitis B

• Hepatitis C

The principle of ELISA is to assay a reaction which uses an enzyme which can be easily assayed with an antigen or specific antibodies. Thus, it involves an antibody and an enzyme which gives a colour change reaction which is a diagnostic test for diseases detection. The concentration of antigens or specific antibody can be determined by ELISA.

Yes, some of the diseases can be detected before its symptoms like using PCR (Polymerase chain reaction) which is used to amplify a nucleic acid to obtain an amplified gene product which can show diseases before its symptoms.

Another method for detection of a disease before the symptoms is ELISA (i.e., Enzyme-Linked Immunosorbent Assay) which can give the concentration of antigens or a specific antibody, which can be used for detection of disease before its symptoms.

The term biopiracy refers to the unethical and unauthorized use of biological resources by multinational companies and other organizations without permission from concerned authorities and individuals.

For example, the fungicidal properties of Neem were patented by an American Multinational company while India has used properties of neem since ancient times. Thus, the American Multinational the company had bio-pirated the knowledge of fungicidal properties of Neem from the Indians without giving due credits or recognition.

The mechanism is useful for micro-organisms because they become active only by certain metabolic and biochemical changes like pH changes or temperature changes. This makes these toxins resistant to protein and the micro-organisms do not get killed by the host's protein action.

Not respecting the genetic barriers can be dangerous because:

• It could lead to the production of unwanted organisms which can affect and disrupt the general food chain of other organisms.

• Superweeds might develop as these GMOs which are used as weedicides can reproduce and itself become a super weed and destroy crops.

• Changes in gene expression by genetic modification can lead to adverse irreversible changes in organisms.

Due to following reason:

• It prevents overexploitation of bio-resources and the use of ancient Indian knowledge and techniques by other countries.

• Second amendment set the rule for patent rights.

• Basmati rice variety belongs to India and cultivated since the time immemorial.

• The patent crop claimed by American company was actually derived by crossing the Indian rice variety.

It was obtained from the pancreas of slaughtered animals.

It developed allergy in humans due to slight changes in polypeptide chain.

• Transgenic animals are used as modle organism for the production of desired products.

• They are used to study diseases like cancer, Alzhimer and their treatment.

• In future it can be used to grow human organs for transplant purposes.

Rosie was the first transgenic cow. Gene coding for the production of α-lactoalbumin was introduced into the cow.

PCR involves the amplification of DNA strands. Hence, infectious organism can be detected at early stage of infection due to production of its multiple copies which can be detected easily.

Following are the objectives:

• GEAC makes the decision regarding the validity of genetic recombination.

• To test the safety of introduction of Genetically modified organism in public services.

Patent was filled for a rice variety obtained by crossing Basmati rice and semi dwarf variety of rice.

Advantages of GMO-

• GM plants have increase nutritional value and higher yield.

• These are cost effective due to increased yield.

• Genetic engineering increases resistance toward pests and diseases e.g. Bt cotton.

• Transgenic animals are used for the production of desired products like nutritional value of milk increased due to production of alpha-lacto albumin.

Gene expression can be controlled with the help of RNA by a method known as RNA interference (i.e., RNAi) in which a gene is “knocked out’’ and the cellular mechanism is degraded. It involves the gene silencing of a specific mRNA which is done by binding a complementary double-stranded RNA (i.e., dsRNA) to the mRNA which inhibits the translation process.

It inhibits the viral antigen gene expression. For example: protection from nematode infection using RNA interference. The Agrobacterium vector forms a complementary dsRNA with the nematode genes and inhibits translation and the nematode infection gene expression doesn’t occur.

Countries like India are underdeveloped countries which are very rich in biodiversity as compared to developed countries like America. These developed countries often under compensate and un-credit the underdeveloped countries for their traditional knowledge regarding the biodiversity of various plants and other organisms.

Thus, if countries like India don’t have the proper biological patenting rules for its traditional knowledge then thedeveloped countries would keep on exploiting its resources without giving any due credit or compensation.

Genetic modification of plants has been very useful in the following areas:

i) Genetically modified plants are more resistant towards abiotic stresses like climate, temperature and pH changes.

ii) Genetically modified plants can give more product yield. GM crops can produce higher yield of food crops. And it also increases the shelf-life of fruits and vegetables.

iii) Genetically modified plants are more resistant towards pests and insects which help in the production of better pest resistant crops.

iv) Genetically modified plants are used to grow crops with higher nutritional values like starch, proteins, vitamins.

Recombinant DNA vaccines are the vaccines which are manufactured by Recombinant DNA technology. It involves the construction of a plasmid using a copy of DNA from pathogen with an empty plasmid.

Examples:

i) Hepatitis B produced from yeast culture (Saccharomyces cerevisiae).

ii) Japanese encephalitis vaccine.

The line of treatment for a genetic disease is different from infectious diseases as the genetic disease requires a change or alteration in the gene instead of any part or organ. Thus, genetic diseases require treatment of the genome by methods like gene therapy which is generally carried out at the embryo stage or early infant stages. These genetic diseases are not caused by any pathogens unlike infectious diseases and therefore, they require a different line of treatment.

A probe is used in molecular diagnostics for labelling a gene or small fragment of genome segment. It is a RNA or DNA fragment which is used for labelling DNA sequences and for genomic mapping and sequencing. A probe is generally radio-labelled or fluorescent labelled and detected by various methods.

A probe is taken and it forms a complementary DNA with the normal (template) DNA but doesn’t attach to mutated DNA. Thus, it can be used for labelling of the mutated gene for molecular diagnostics.

The first patient who was given gene therapy was a four-year-old with adenosine deaminase (ADA) deficiency. The adenosine deaminase (ADA) deficiency is cured by growing lymphocytes outside the body of the patient in a cell culture and using a functional ADA complementary DNA (i.e., cDNA) in those lymphocytes. The treatment is recurrent because these ADA cDNA are not immortal.

Thus, this treatment is required to be repeated for new cells and cure the adenosine deaminase (ADA) deficiency.

Upstream processing is the method and techniques which are involved in the isolation of DNA. It is used for isolating useful gene products. It involves using restriction endonucleases and screening of cells for its transformation. An example of upstream processing is used in the production of Insulin using rDNA technology.

Downstream processing is the separation, purification, quality control and clinical trials of a product before commercially selling it. Various separation and purification techniques are used like ultrafiltration, chemical degradation, etc. Example of downstream processing is the production of Insulin using rDNA technology.

Antigen- Any foreign body which can stimulate antibody formation by immune system after entering the body

Antibody- it is the glycoprotein produced by the immune system foe defence purposes.

Two digonostic tests based upon antigen- antibody relationship are-

a. ELISA for AIDS

b. Pregnancy test

Phenylketonuria is an in born error which occurs due to absence of enzyme required for the conversion of tyrosine into phenylalanine.

Yes, ELISA can be used to detect the presence of phenylketonuria. The patient in which enzyme-antibody complex enzyme is absent will give negative test, and would prove the absence of enzyme. Hence positive test for phenylketonuria.

- Pro insulin contains 3 chains of polypeptides i.e. A, B & chain C.

Chain A have 21 amino acids, B contains 30 amino acids & chain c consists of 33 amino acids.

When C-peptide is removed and cut, A & B peptide chains are joined by disulfide bonds, mature, functional insulin is formed.

- Enzyme replacement therapy is not a permanent method to cure the disease as it is needed to be given at regular intervals to the patient.

- Due to the potential to completely cure the genetic defect, gene therapy is considered to be a better option.

If, gene replacement is done during embryonic condition it could be a permanent cure

Animals in which a foreign of DNA is incorporated so as to obtain desired products or to study its effects on the biological processes are called the transgenic animals.

- E.g. – in 1997, Rosie a cow has been made transgenic to produce α-lactalbumin rich milk (2.4g/L).

Animal models have been developed to provide α-1-antitrypsin used by humans for the treatment of emphysema.

- In Canada, gene for spider web has been isolated from spiders & was inserted into goat. The goat now produces spider webs along with milk.

Due to the great tensile strength & light weight these spider web fibres are used for the development of military uniforms & tennis rackets.

- The foreign DNA sequence is joined with the vectors plasmid using DNA ligase.

- Whenever the cell replicates its DNA ultimately leads to the replication of foreign DNA sequence.

- Form on generation to other generation along the vector’s DNA the foreign DNA is also passed on.

Bt toxin is a species specific, narrow spectrum biopesticide. It can only provide protection to the plant only from the insects belonging to lepidopteron, dipterans and coleopterans.

ADA (i.e., adenosine deaminase) deficiency is a genetic disorder caused by the deletion of a gene for adenosine deaminase synthesis which leads to the deficiency of the ADA enzyme in the body which causes damage to the immune system. It can be cured by various approaches like:

i) A bone marrow transplant can cure the disease. Suitable bone marrow is required for the transplant.

ii) Enzyme replacement therapy can be used. The adenosine deaminase enzyme can be injected in the body.

Both of these approaches help in curing ADA but they do not completely cure the ADA deficiency disease.

Another approach of gene therapy has been found to be highly successful. It involves growing lymphocytes outside the body of the patient in a cell culture and using a functional ADA complementary DNA (i.e., cDNA) in those lymphocytes. This treatment is recurrent in nature because these ADA cDNA are not immortal and new cells need to be injected timely to the patient however if this procedure can be carried at an early embryonic stage in the bone marrow then gene therapy can be a permanent cure.

Transgenic animals are the animals which are genetically modified for various purposes. They contain a ‘transgene’ which is a foreign gene which is inserted into the target organism by various recombinant DNA technology methods to bring modifications in their gene expression.

Transgenic animals are utilized in the following four areas:

i) Biological studies: These animals are studied by various biologists and researchers to understand the physiology of animals and to understand how gene expression affects these animals.

ii) Increases yield product: Transgenic animals like cows and sheep have been modified to yield more milk or give better wool quality.

iii) Testing and clinical trials: Transgenic organisms like transgenic mice have been used widely for clinical trials and vaccine testing before the human trials.

iv) Diseases study and chemical safety testing: Transgenic animals are often used as biological models for understanding diseases like cancer and Parkinson’s disease and they are also used for testing the toxicity of a chemical. They give faster results without violating any human ethics.

The above steps are needed to be followed sequentially to perform a gene transfer to a plant and obtain a transgenic plant.

The five areas where biotechnology has influenced our lives are:

i) Therapeutics and Diagnostic tool: Biotechnological methods like PCR (i.e., Polymerase Chain Reaction) is widely used as a diagnostic tool for disease detection and methods like gene therapy are used for treating genetic diseases like adenosine deaminase deficiency and rDNA technology is used for the production of Insulin and Human Growth Hormone (HgH).

ii) Genetically modified plants and animals: The use of genetically modified organisms has produced more healthy and nutritional crop yields and transgenic animals are used to get more products like milk from cows, wool from sheep and alcohol from yeasts etc.

iii) Forensic and paternity tests: Biotechnological methods like DNA fingerprinting has helped in solving crime cases and determination of genetic parents.

iv) Wastewater treatment: Biotechnology has been widely used in the wastewater treatment plants, Micro-organisms are added with the wastewater which is digested by the bacteria and it helps in the degradation of organic waste.

v) Energy production: Biotechnology is used for the production of energy through micro-organisms and producing bio-fuel. cellulose enzyme technology is used to convert crop residues to ethanol.

The various advantages of using genetically modified plants to increase the overall yield of the crop are:

i) Genetically modified plants are more resistant towards abiotic stresses like climate, temperature and pH changes. Thus, the plants are able to grow normally in unfavourable conditions.

ii) Genetically modified plants can give more yield of products. GM crops has proved to produce higher food crop yield as compared to normal crops.

iii) Genetically modified plants are more resistant towards pests and insects which helps in producing better crops which are disease-resistant.

iv) Genetically modified plants are used for growing crops with higher nutritional values in starch, proteins, vitamins, etc. Thus, these crops are commercially more preferred due to their increased nutritional value.

v) Genetically modified plants have products i.e., fruits or vegetables with a much longer shelf life. Thus, these plants can be used to produce fruits or vegetables which can be kept for a longer period of time before they start to degrade and rot.

(a) A nematode Meloidogyne incognita is known to cause root rot in tobacco plant

- Initially nematocides were used to deal with this nematode.

- But later on, the scientists modified the tobacco crop and adopted a method to kill the nematode called the RNA interference (RNAi).

- In the RNAi mechanism, scientist introduced a host specific double standard DNA using Agrobacterium tumefaciens.

- The DNA had both sense & antisense sequences so, that when transcription happen complementary RNA strands of both sense & antisense strands are produced.

- Both RNA strands being complementary forms double strands & gets chop-off since RNA of nematode gets inactivate.

(b) Golden rice is the example of genetically modified crops.

- β-carotene gene from daffodil plant was introduced into the rice plant, in order to nutritionally enrich it with vitamin A.

- β-carotene is the precursor of vitamin A.

Disadvantages are as follows-

- The insulin obtained from slaughtered animals was slightly different from humulin and caused allergic reactions in humans.

- Slaughtering animals for the insulin is against ethics.

- Cost of bovine insulin was very high.

- Recombinant insulin had no allergic reactions, while bovine caused allergy.

- This stopped slaughtered of large number of animals.

Supply is many folds higher which resulted in comparatively low cost as compared with bovine insulin.

- Bio-pesticides are the biologically present substances which are used to control the pests.

- These are narrow spectrum & species specific.

- e.g. – Bt toxin

- Bt toxin obtained from Bacillus thuringiensis is used against the insects.

- The insects when ingest Bt toxin, due to presence of alkaline nature Bt toxin gets activate, binds to the gut wall, creates pores hence the insects die.

The 5 tools for rDNA technology are as follows

a) Restriction endonuclease - It cuts the DNA sequence at specific positions.

b) Vector - It is the most important tool for the rDNA technology vector contains site for the insertion of gene of interest called the origin of replication.

c) DNA ligase - It facilitates the joining of desired DNA with vector DNA.

d) Polymerase - The enzyme makes multiple copies of rDNA.

e) Host cell - The cell which takes up rDNA & change itself to transformant.