Question 1

What do you mean by DNA fingerprinting? Explain it through RFLP.

Accepted Answer

DNA fingerprinting is a technique used to identify individuals based on unique patterns in their DNA. It involves analyzing specific regions of DNA that vary highly among individuals. Restriction Fragment Length Polymorphism (RFLP) is one method used in DNA fingerprinting. In RFLP, DNA is cut into fragments using restriction enzymes. These fragments are then separated by gel electrophoresis. The pattern of fragments is unique for each individual due to variations in DNA sequences at restriction sites. By comparing these patterns, individuals can be identified or relatedness can be established. — Step 1: Extract DNA from the sample.
Step 2: Use restriction enzymes to cut DNA at specific sequences.
Step 3: Separate the resulting fragments by gel electrophoresis.
Step 4: Transfer DNA fragments to a membrane (Southern blotting).
Step 5: Hybridize with a labeled DNA probe complementary to a specific sequence.
Step 6: Visualize the pattern of bands. Differences in band patterns among individuals constitute the DNA fingerprint.

Question 2

What are GMOs? Describe the method of development of transgenic plants.

Accepted Answer

GMOs (Genetically Modified Organisms) are organisms whose genetic material has been altered using recombinant DNA technology to express desired traits. Transgenic plants are developed by introducing foreign genes into their genome to confer beneficial traits such as pest resistance or herbicide tolerance.

Method of development of transgenic plants:
1. Identification and isolation of the desired gene.
2. Insertion of the gene into a suitable vector (e.g., Ti plasmid).
3. Introduction of the vector into plant cells by methods such as Agrobacterium-mediated transformation or gene gun.
4. Selection of transformed cells using selectable markers.
5. Regeneration of whole plants from transformed cells through tissue culture techniques.
6. Screening and confirmation of transgenic plants expressing the desired trait. — The process involves molecular cloning of the gene of interest, vector construction, transformation of plant cells, and regeneration of plants. Agrobacterium tumefaciens is commonly used for dicot plants, while gene gun is used for monocots. Selectable markers like antibiotic resistance help identify transformed cells.

Question 3

Differentiate between direct and indirect method of gene transfer. Name one indirect method suitable for gene transfer in dicot plants.

Accepted Answer

Direct method of gene transfer involves the direct introduction of foreign DNA into the plant cells without the use of any intermediary organism. Examples include gene gun (biolistics) and electroporation.

Indirect method involves the use of a vector, usually a microorganism, to transfer the gene into the plant cells. The most common indirect method is Agrobacterium-mediated transformation.

Difference:
- Direct method does not require a vector; indirect method uses a vector.
- Direct method is physical or chemical; indirect method is biological.
- Direct method is often used for monocots; indirect method is suitable for dicots.

One indirect method suitable for gene transfer in dicot plants is Agrobacterium tumefaciens-mediated transformation. — Agrobacterium tumefaciens naturally infects dicot plants and transfers part of its Ti plasmid DNA into the plant genome, making it an effective vector for gene transfer in dicots.

Question 4

What is molecular pharming? Give applications of transgenic animals in molecular pharming.

Accepted Answer

Molecular pharming is the use of genetically modified plants or animals to produce pharmaceutical substances such as vaccines, antibodies, or therapeutic proteins.

Applications of transgenic animals in molecular pharming:
- Production of human therapeutic proteins like insulin, growth hormones, and clotting factors in milk, blood, or eggs.
- Production of monoclonal antibodies for disease treatment.
- Generation of animals that produce vaccines or other biologically active molecules.
- Use in gene therapy research and development. — Transgenic animals are engineered to carry and express human genes that code for valuable proteins. These proteins can be harvested from animal secretions, providing a cost-effective and scalable method for producing pharmaceuticals.

Question 5

Differentiate between gene gun and gene therapy.

Accepted Answer

Gene gun is a physical method used to introduce foreign DNA into plant cells by bombarding them with DNA-coated microscopic particles. It is mainly used in plant genetic engineering.

Gene therapy is a medical technique used to treat genetic disorders by introducing, removing, or altering genetic material within a patient's cells.

Differences:
- Gene gun is a tool for gene transfer in plants; gene therapy is a treatment method in humans.
- Gene gun delivers DNA physically; gene therapy may use viral or non-viral vectors.
- Gene gun is used for crop improvement; gene therapy is used for disease treatment. — Gene gun is a laboratory technique for plant transformation, while gene therapy is a clinical approach to correct defective genes in patients.

Question 6

Give the procedure of development of recombinant subunit vaccines.

Accepted Answer

Procedure for development of recombinant subunit vaccines:
1. Identify and isolate the gene encoding the antigenic protein of the pathogen.
2. Clone the gene into an expression vector.
3. Introduce the vector into a suitable host (bacteria, yeast, or mammalian cells) to produce the antigenic protein.
4. Purify the expressed protein.
5. Formulate the purified protein with adjuvants to enhance immune response.
6. Test the vaccine for safety and efficacy before use. — Recombinant subunit vaccines use only the antigenic parts of the pathogen, reducing the risk of infection. The recombinant protein stimulates the immune system to produce immunity.

Question 7

Write a short note on DNA vaccines.

Accepted Answer

DNA vaccines involve the direct introduction of a plasmid containing the gene encoding an antigen into the host cells. The host cells then produce the antigen, stimulating an immune response. DNA vaccines are stable, easy to produce, and can induce both humoral and cellular immunity. — Unlike traditional vaccines, DNA vaccines do not use live or killed pathogens but rely on the host's cellular machinery to produce the antigen.

Question 8

Describe the advantages of monoclonal antibodies developed by rDNA technology over that developed by Hybridoma technology.

Accepted Answer

Advantages of monoclonal antibodies developed by rDNA technology over hybridoma technology include:
- Ability to produce humanized or fully human antibodies, reducing immunogenicity in patients.
- Large-scale production is easier and more consistent.
- Avoids the use of animals and hybridoma cell lines.
- Genetic engineering allows modification of antibody properties such as affinity and specificity.
- Faster production and reduced cost. — Hybridoma technology produces monoclonal antibodies by fusing B cells with myeloma cells, which may produce murine antibodies that can cause immune reactions in humans. rDNA technology enables the production of antibodies better suited for therapeutic use.

Question 9

Briefly describe the development of Humulin through rDNA technology.

Accepted Answer

Humulin is genetically engineered human insulin produced by recombinant DNA technology. The human insulin gene is cloned into a plasmid vector and introduced into Escherichia coli bacteria. The bacteria express the insulin protein, which is then purified and processed to produce biologically active insulin identical to human insulin. This method provides a reliable and safe source of insulin for diabetic patients. — The process involves isolating the insulin gene, cloning into bacterial plasmids, transforming E. coli, inducing protein expression, and purification of insulin. This replaced the earlier use of animal insulin.

Question 10

Write a short note on humatrope and Protropin.

Accepted Answer

Humatrope and Protropin are recombinant human growth hormones produced by rDNA technology. They are used to treat growth hormone deficiency and related disorders. These synthetic hormones are identical to natural human growth hormone and help in promoting growth and development. — These hormones are produced by inserting the human growth hormone gene into bacterial or mammalian cells, which then produce the hormone for therapeutic use.

Question 11

Briefly describe the applications of rDNA technology in crop improvement.

Accepted Answer

Applications of rDNA technology in crop improvement include:
- Development of pest-resistant crops by introducing Bt genes.
- Herbicide-resistant crops allowing selective weed control.
- Improvement of nutritional quality, e.g., Golden Rice enriched with Vitamin A.
- Development of disease-resistant plants.
- Enhanced tolerance to abiotic stresses like drought and salinity.
- Increased yield and shelf life of crops. — Genetic engineering allows precise introduction of beneficial traits into crops, improving productivity and sustainability.

Question 12

List the ethical issues related to the use of transgenic animals?

Accepted Answer

Ethical issues related to the use of transgenic animals include:
- Animal welfare concerns due to genetic modifications.
- Potential ecological impacts if transgenic animals escape into the wild.
- Moral concerns about 'playing God' and altering natural organisms.
- Issues related to patenting and ownership of genetically modified animals.
- Possible risks to human health from consumption or use of products derived from transgenic animals. — These concerns require careful regulation and ethical review before the use of transgenic animals in research or industry.

Question 13

What is the role of vaccinia virus in the development of recombinant vaccine?

Accepted Answer

Vaccinia virus is used as a vector in recombinant vaccine development. It can carry and express foreign genes encoding antigens from other pathogens. When used as a vaccine, it stimulates the immune system to recognize and respond to these antigens, providing immunity without causing disease. — Vaccinia virus is safe and effective as a carrier because it can infect human cells and express inserted genes, making it useful for recombinant vaccine production.

Question 14

Write a short note on recombinant therapeutic agents.

Accepted Answer

Recombinant therapeutic agents are proteins or molecules produced using recombinant DNA technology for medical treatment. Examples include insulin, growth hormones, clotting factors, and monoclonal antibodies. These agents are used to treat various diseases and conditions with high specificity and efficacy. — They are produced by cloning the gene of interest into host cells, which then express the therapeutic protein for purification and use.

Question 15

Write a short note on humanised antibodies.

Accepted Answer

Humanised antibodies are monoclonal antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. This reduces the immune response against the antibody when used as a therapeutic agent. — Humanisation involves replacing most of the murine antibody sequence with human antibody sequences except the antigen-binding sites, improving compatibility and reducing side effects.

Applications of Recombinant DNA Technology

Applications of Recombinant DNA Technology — Study Notes

Introduction

Production of Human Insulin

Production of Vaccines

Practice Questions — Applications of Recombinant DNA Technology

All 13 Chapters in Biotechnology