Animal Biotechnology 2019
- Answer any two questions from the following:
a) Schematically describe the process of successful cloning in sheep. Name two therapeutic proteins that can be produced in the milk of domestic animals. (3+2=5)
Ans→1) Selection of Donor Cells: Somatic cells (e.g., mammary gland cells) are collected from a donor sheep and cultured.
2) Preparation of Egg Cell: An unfertilized egg is taken from a different sheep, and its nucleus (containing the genetic material) is removed, creating an enucleated egg.
3) Nuclear Transfer: The nucleus of the donor somatic cell is inserted into the enucleated egg. This reprograms the somatic cell’s DNA to behave like an embryonic cell.
4) Activation of Egg: The reconstructed egg is stimulated using an electric pulse to initiate cell division, mimicking fertilization.
5) Embryo Development: The activated egg begins to divide and develop into an embryo under laboratory conditions.
6) Embryo Implantation: The developing embryo is implanted into the uterus of a surrogate sheep.
7) Birth of Clone: After a normal gestation period, the surrogate sheep gives birth to a lamb (clone) genetically identical to the donor sheep.
Alpha-1-antitrypsin(A1AT) and monoclonal antibodies, tPA are the two proteins that are produced by domestic animals.
b) What do you mean by a DNA library? Schematically describe the making of a genomic library. (1+4=5)
Ans→ A DNA library is a collection of DNA fragments that represent the genetic material of an organism used for studying genes, sequencing, and functional analysis.
DNA Isolation → 2. Fragmentation of DNA → 3. Vector Preparation → 4. Ligation → 5. Transformation → 6. Screening and Storage
- Isolation of Genomic DNA
Extract DNA from the organism’s cells using chemical or enzymatic methods. Purify the DNA to remove contaminants.
- Fragmentation of Genomic DNA
The genomic DNA is fragmented into smaller pieces using restriction enzymes or mechanical shearing. These fragments may have overlapping regions to ensure full coverage.
- Vector Preparation
Plasmid or phage vectors are cut using the same restriction enzyme used to fragment the genomic DNA. The vector is linearized and ready for insertion of DNA fragments.
- Ligation
The DNA fragments are ligated into the prepared vectors using DNA ligase. Each vector receives a unique DNA fragment.
- Transformation
The recombinant DNA (vector + insert) is introduced into a host organism (e.g., E. coli) using transformation techniques like heat shock or electroporation. Each host cell takes up a single vector containing one DNA fragment.
- Screening and Storage
Host cells are grown on selective media to identify those containing recombinant DNA.
c) Diagrammatically describe the steps of DNA fingerprinting as used for a paternity case. (5)
Ans→ DNA fingerprinting in Paternity Case
- Sample Collection: Obtain DNA samples from the alleged father and child using buccal swabs on blood samples.
- Isolation of DNA: Extract DNA from the collected samples using specialized techniques to seperate the genetic material from other cellular components.
- PCR Amplification: Replicate multiple copies of specific regions of the DNA through PCR technique.
- DNA fragmentation: The amplified DNA is fragmented by the restriction enzymes.
- Gel Electrophoresis: Separate the DNA fragments based on size by gel electrophoresis.
- Southern Blotting: Transfer the separated DNA fragment from the gel to a membrane.
- Hybridization: Adding labelled DNA probes that bind specifically to the target DNA sequences, labeling them for detection.
- Autoradiography: Visualize the labelled DNA fragments through autoradiography revealing a unique pattern of bands for each individual (son, father)
- Analysis and Comparison: Compare the DNA band patterns of the alleged father and child. Probability of paternity based on the DNA pattern, considering the prevalence of biological relationship
Animal Biotechnology 2020
- Answer any two questions from the following: 5×2=10
a) Schematically describe the making of cDNA library. What is plaque hybridization? (4+1)
Ans→ Process of Making a cDNA Library:
- mRNA Isolation
Extract mRNA from the cells or tissue of interest.
↓
- First-Strand cDNA Synthesis
Use reverse transcriptase and an oligo(dT) primer to synthesize the first cDNA strand using mRNA as a template.
↓
- Second-Strand cDNA Synthesis
RNase H removes RNA fragments, and DNA polymerase synthesizes the second cDNA strand.
↓
- Addition of Linkers/Adapters
Attach synthetic linkers or adapters containing restriction sites to the ends of the cDNA.
↓
- Cloning into Vectors
Insert cDNA fragments into prepared vectors using restriction enzymes and DNA ligase.
↓
- Transformation into Host Cells
Introduce recombinant vectors into competent host cells (e.g., E. coli).
↓
- Screening and Storage
Identify colonies containing cDNA inserts and store the cDNA library in microplates or as frozen stocks.
Plaque hybridization is a technique used to identify specific DNA sequences in bacteriophage plaques using labeled probes that hybridize to the target sequence.
b)What do you mean by cloning vectors? Briefly discuss about bacteriophage Lambda and expression vectors. (1+2+2)
Ans→ Cloning vectors are DNA molecules, used to carry foreign DNA into host cells for replication and expression.
Bacteriophage Lambda:
Bacteriophage lambda is a virus that infects Escherichia coli and is widely used in molecular biology as a cloning vector. Its genome can integrate into the bacterial chromosome (lysogenic cycle) or exist as a lytic phage. Lambda vectors are modified to carry foreign DNA by replacing non-essential genes, allowing efficient cloning of large DNA fragments (up to 25 kb).
Expression Vectors:
Expression vectors are specialized cloning vectors designed for protein production. They contain regulatory elements like promoters, ribosome binding sites, and terminators to drive high-level expression of the inserted gene in host cells. These vectors are used to produce proteins for research, medicine, and industry. Examples include plasmids and bacteriophage-derived vectors.
c)Briefly describe the production method of transgenic animals using DNA microinjection with suitable diagrams. Write two applications of transgenic animals. (3+2)
Ans→ Method of Production of Transgenic Animals using DNA Microinjection
- Desired gene isolation: Isolate the gene of interest from an organism.
- Plasmid Construction: Insert the gene into a plasmid creating a recombinant DNA molecule.
- Microinjection: Inject the recombinant DNA or gene of interest into the pronucleus of a fertilized egg. This is usually done at the single cell stage.
- Implantation: Transfer the injected embryos into the reproductive tract of a surrogate mother.
- Growth and Development: Allow the embryos to develop into transgenic animals within the mother.
- Genetic Screening: Confirm the presence of desired gene in the offspring through genetic screening.
Application of Transgenic Animals:
- 1) Transgenic Animals are developed for production of pharmaceuticals to cure diseases.
- 2) They are design to express desire trade such as resistance to disease enhanced nutritional content in their product (e.g milk meet).
Animal Biotechnology 2021
a) Describe the steps of DNA fingerprinting as used for a paternity case.
Ans→ DNA fingerprinting in Paternity Case
- Sample Collection: Obtain DNA samples from the alleged father and child using buccal swabs on blood samples.
- Isolation of DNA: Extract DNA from the collected samples using specialized techniques to seperate the genetic material from other cellular components.
- PCR Amplification: Replicate multiple copies of specific regions of the DNA through PCR technique.
- DNA fragmentation: The amplified DNA is fragmented by the restriction enzymes.
- Gel Electrophoresis: Separate the DNA fragments based on size by gel electrophoresis.
- Southern Blotting: Transfer the separated DNA fragment from the gel to a membrane.
- Hybridization: Adding labelled DNA probes that bind specifically to the target DNA sequences, labeling them for detection.
- Autoradiography: Visualize the labelled DNA fragments through autoradiography revealing a unique pattern of bands for each individual (son, father)
- Analysis and Comparison: Compare the DNA band patterns of the alleged father and child. Probability of paternity based on the DNA pattern, considering the prevalence of biological relationship.
b) What do you mean by contact inhibition? Describe different phases in the growth pattern. of an established all line. (2+3)
Ans→ Contact inhibition refers to the phenomenon where cells stop dividing when they come into contact with neighbouring cells, preventing overcrowding and maintaining tissue homeostasis.
Different phases of growth of cell line.
- Lag phase:
- Initial period of adjustment.
- Cells adapt to new environment and conditions.
- Limited or no growth during this phase.
- Log or Exponential phase
- Rapid and exponential cell growth.
- Cells actively divide and population increases dramatically.
- Conditions are favorable for growth.
- Stationary Phase:
- Growth rate slows down.
- Cell division and death reach a balance.
- Limited resources lead to competition among cells.
- Death Phase:
- Cell death exceeds cell division
- Depletion of nutrients and accumulation of waste products.
- Population declines.
- Long term Stationary Phase:
- Some cells may enter a prolonged, non dividing state.
- Population stabilizes at a lower level.
- Survival strategies are employed by remaining cells.
c) What do you mean by cDNA Library? Show the diagramatic steps in the formation of double stranded cDNA from an mRNA. (1+4)
Animal Biotechnology 2022
a) Schematically describe the process of successful cloning in sheep. Name two therapeutic proteins that can be produced in the milk of domestic animals. (3+2)
Ans→1) Selection of Donor Cells: Somatic cells (e.g., mammary gland cells) are collected from a donor sheep and cultured.
2) Preparation of Egg Cell: An unfertilized egg is taken from a different sheep, and its nucleus (containing the genetic material) is removed, creating an enucleated egg.
3) Nuclear Transfer: The nucleus of the donor somatic cell is inserted into the enucleated egg. This reprograms the somatic cell’s DNA to behave like an embryonic cell.
4) Activation of Egg: The reconstructed egg is stimulated using an electric pulse to initiate cell division, mimicking fertilization.
5) Embryo Development: The activated egg begins to divide and develop into an embryo under laboratory conditions.
6) Embryo Implantation: The developing embryo is implanted into the uterus of a surrogate sheep.
7) Birth of Clone: After a normal gestation period, the surrogate sheep gives birth to a lamb (clone) genetically identical to the donor sheep.
Alpha-1-antitrypsin(A1AT) and monoclonal antibodies, tPA are the two proteins that are produced by domestic animals.
b) Briefly describe the production method of transgenic animals using DNA microinjection with suitable diagrams. Write two applications of transgenic animals. (3+2)
Ans→
c) What do you mean by gene therapy? How it is useful for Human disease cure? (2+3)
Ans→ Gene therapy is a medical approach that involves introducing, altering or replacing genetic material within a person’s cells to treat or prevent diseases.
Ans→ Gene therapy treating human diseases by correcting or replacing faculty genes. The goal is to restore normal cellular function and alleviate symptoms by addressing the underlying genetic causes of the diseases.
① Correcting Genetic Defects: Gene therapy can replace or repair faulty genes responsible for genetic disorders addressing the root cause of the disease.
②Treating Genetic and Acquired disease: Effective for both inherited and acquired disease by modifying the patient’s genetic makeup.
③ Enhancing Immune Response: Boosting the immune system by introducing genes enhance the body’s ability to fight infections that on cancer.
④ Precision Medicine: Tailored treatments based on individual genetic make up for enhanced efficacy and reduced side effects.
⑤ Disease Prevention: By introducing healthy genes it can prevent the development of disease with a genetic basis.