2022 —

Time :As in Programme ;
FullMarks:60 |
The figures in the right-hand margin indicate marks.
Draw labelled diagram wherever necessary
Answer all questions.
Part — I

Answer the following questions (Fill i in ‘the blanks/ One word
answer) 1x8

a. EcoRI is a restriction enzyme that produces sticky ends.

b. Southern hybridization was developed by Edward Southern.

c. EcoRV is an example of a blunt end cutter enzyme.

d. cDNA stands for complementary DNA.

e. The vector most commonly used for plant gene transfer is Agrobacterium tumefaciens.

f. Genetic engineering is also known as recombinant DNA technology.

g. DNA fragments are ligated with the help of the enzyme DNA ligase.

h. The PCR technique was invented by Kary Mullis.

Define transformation: Transformation is the process by which a cell takes up foreign DNA from its environment, incorporating it into its own genome. This can lead to a change in the cell's genotype and potentially its phenotype.

What is an episome?: An episome is a type of plasmid in bacteria that can exist either as an independent plasmid or integrate into the bacterial chromosome. It carries genetic material that can be passed on during cell division, and often plays a role in antibiotic resistance or virulence.

Write the function of reverse transcriptase enzyme: Reverse transcriptase is an enzyme that catalyzes the conversion of RNA into complementary DNA (cDNA). This is a crucial process in retroviruses and is used in laboratory techniques to create cDNA libraries from mRNA.

What is T-DNA?: T-DNA (Transfer DNA) is the segment of DNA from the Ti plasmid of Agrobacterium tumefaciens that is transferred into the plant genome during genetic transformation. T-DNA contains genes that can cause tumor formation or be used for gene transfer in genetic engineering.

Define Genome Mapping: Genome mapping refers to the process of determining the location and sequence of genes within a genome. It helps identify the physical position of genes and their relative distance from one another, which is important for understanding genetic traits and disease.

Function of selectable marker: A selectable marker is a gene introduced into an organism to enable the identification of cells that have successfully incorporated foreign DNA. It typically confers resistance to antibiotics or other selective agents, allowing transformed cells to be isolated.

Write the function of Taq DNA polymerase: Taq DNA polymerase is a heat-stable enzyme derived from the bacterium Thermus aquaticus. It is used in the polymerase chain reaction (PCR) to amplify DNA because it can withstand the high temperatures required for DNA denaturation.

Which bacterium is used for the production of insulin by genetic engineering?: The bacterium Escherichia coli is commonly used for the production of insulin through genetic engineering. Human insulin genes are inserted into E. coli, which then produces insulin that can be harvested and used in medical treatments.

What is a Probe?: A probe is a fragment of DNA or RNA that is labeled with a detectable marker and used to detect the presence of complementary sequences in a sample. Probes are widely used in techniques like Southern blotting and in situ hybridization.

What is an immunomodulator?: An immunomodulator is a substance that can modify or regulate the immune system. It can either enhance or suppress immune responses, depending on the type of immunomodulator used. They are often used to treat immune-related disorders or in cancer immunotherapy.

 Part-III

Answer any eight questions

Part-IV

4. Answer thé followings

1. Discuss the types of cloning vectors used in genetic engineering.

Cloning vectors are DNA molecules that are used to carry foreign genetic material into a host cell for replication or expression. Various types of cloning vectors are used depending on the nature of the cloning experiment and the host cell being used. The main types of cloning vectors used in genetic engineering are:

• Plasmid Vectors: These are the most commonly used cloning vectors, particularly in bacterial systems. Plasmids are small, circular DNA molecules found in bacteria and can replicate independently of chromosomal DNA. They are useful for cloning small to medium-sized DNA fragments. Common examples of plasmid vectors include pUC18, pBR322, and pGEM.

• Bacteriophage Vectors: Bacteriophages (viruses that infect bacteria) can be used as cloning vectors. These vectors are capable of infecting bacterial cells and integrating foreign DNA into the bacteriophage genome. Examples include λ phage vectors, which can carry larger DNA inserts compared to plasmids.

• Cosmid Vectors: Cosmids are hybrids between plasmids and bacteriophages. They can carry larger DNA inserts (up to 45 kb) than plasmids. Cosmids are especially useful for constructing genomic libraries of large organisms and are typically used in E. coli for cloning.

• Bacterial Artificial Chromosome (BAC) Vectors: BACs are used for cloning large DNA fragments (up to 300 kb). These vectors are based on the F-plasmid, which is responsible for bacterial conjugation. BACs are widely used in genome sequencing projects, such as the Human Genome Project.

• Yeast Artificial Chromosome (YAC) Vectors: YACs are used for cloning very large DNA fragments (up to 1,000 kb) in yeast cells. These vectors are especially useful for cloning eukaryotic DNA and studying the structure and function of large genes.

• Expression Vectors: Expression vectors are designed not only for cloning DNA but also for expressing the cloned gene in the host cell. These vectors contain necessary regulatory elements such as promoters and ribosome-binding sites that enable transcription and translation in the host. Examples include pET vectors for bacterial expression and pCMV vectors for mammalian expression.

Each type of cloning vector has specific advantages depending on the size of the DNA insert, the host organism, and the purpose of the cloning experiment. Cloning vectors have revolutionized genetic engineering by making it easier to manipulate and study genes in various organisms.

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2. Write the principle and steps involved in polymerase chain reaction with its applications in Genetic engineering.

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify a specific DNA segment, producing millions of copies from a small DNA sample. PCR has revolutionized genetic research, diagnostics, and biotechnology applications. The principle and steps involved in PCR are as follows:

Principle:

The principle of PCR is based on the ability of DNA polymerase to synthesize new DNA strands using a single-stranded template. PCR amplifies a specific region of DNA by using short synthetic oligonucleotide primers that flank the region of interest. The process relies on repeated cycles of denaturation, annealing, and extension to generate large quantities of the target DNA sequence.

Steps in PCR:

1. Denaturation (94–98°C): The double-stranded DNA is heated to a high temperature (usually 94–98°C) to break the hydrogen bonds between complementary bases, separating the DNA into two single strands.

2. Annealing (50–65°C): The reaction temperature is lowered to allow the primers to bind (anneal) to the complementary sequences on the single-stranded template DNA. Two primers are used, one for each strand of the DNA, to define the region to be amplified.

3. Extension (75–80°C): The temperature is increased to the optimal temperature for the DNA polymerase (typically around 75–80°C for Taq polymerase). The polymerase synthesizes a new strand of DNA complementary to the template strand, extending the DNA sequence from the primers.

4. Repeat cycles: The three steps (denaturation, annealing, and extension) are repeated 20-40 times, leading to an exponential amplification of the target DNA region.

Applications in Genetic Engineering:

PCR has many applications in genetic engineering, including:

• Gene Cloning: PCR is used to amplify genes or regions of DNA of interest, which are then inserted into vectors for cloning into host cells.
• Mutagenesis: PCR is used to introduce specific mutations into genes for functional analysis.
• Diagnostics: PCR is used to detect specific DNA sequences, such as in pathogen detection or genetic disease diagnosis.
• Forensic Science: PCR is used for DNA fingerprinting, enabling the identification of individuals based on unique DNA sequences.
• Gene Expression Studies: PCR can be used to measure gene expression by quantifying mRNA levels in a sample, often using reverse transcription (RT-PCR) to convert mRNA into complementary DNA (cDNA).

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3. Describe the procedure for preparation of cDNA library and the significance of cDNA library.

A cDNA library is a collection of complementary DNA (cDNA) molecules that are synthesized from mRNA and represent the expressed genes in a particular cell or tissue at a specific time. cDNA libraries are valuable tools for studying gene expression and identifying genes in specific tissues or under certain conditions.

Procedure for Preparation of cDNA Library:

1. RNA Isolation: Total RNA is first isolated from the target cells or tissues. This RNA sample will contain both mRNA and non-coding RNAs.

2. mRNA Selection: The mRNA is purified from the total RNA using poly(dT) oligonucleotides that bind to the poly-A tails of the mRNA molecules.

3. Reverse Transcription: The purified mRNA is used as a template for reverse transcription. The enzyme reverse transcriptase synthesizes complementary cDNA from the mRNA template using a short oligonucleotide primer (usually poly(dT)).

4. Second Strand Synthesis: The first cDNA strand is then used to synthesize a complementary second strand, often with the help of DNA polymerase, which generates a double-stranded cDNA molecule.

5. Cloning into Vectors: The double-stranded cDNA is ligated into a suitable cloning vector, such as a plasmid or phage vector, and transformed into bacterial cells. This step creates a cDNA library, where each bacterial colony contains a unique cDNA insert.

6. Screening the Library: The cDNA library is screened for specific genes of interest using methods like hybridization or PCR. These genes can then be sequenced, expressed, or analyzed further.

Significance of cDNA Library:

• Gene Expression Studies: cDNA libraries represent the expressed genes in a particular tissue or under specific conditions, making them ideal for studying gene expression profiles.
• Gene Cloning and Functional Studies: The cDNA library can be used to isolate specific genes for functional analysis, allowing researchers to study gene function in vitro or in vivo.
• Identifying New Genes: By screening cDNA libraries, researchers can identify new genes that are expressed in specific tissues or conditions, aiding in gene discovery.
• Functional Genomics: cDNA libraries play a critical role in functional genomics by providing a source of clones for protein production, gene expression studies, and high-throughput screening of gene function.

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4. What is in vitro mutagenesis? Describe in detail the PCR-based method for site-directed mutagenesis.

In vitro mutagenesis refers to the process of introducing specific mutations (such as base substitutions, deletions, or insertions) into a gene or DNA sequence in a controlled laboratory setting. This method is used to study gene function, protein structure, and the effects of mutations on biological processes.

PCR-based Method for Site-Directed Mutagenesis:

One of the most widely used methods for in vitro mutagenesis is the PCR-based method for site-directed mutagenesis. This method involves using PCR to introduce specific mutations into a DNA sequence. The key steps in this method are as follows:

1. Designing Mutagenic Primers: Two primers are designed that are complementary to the target DNA sequence but contain the desired mutation (e.g., a base substitution). These primers are used to amplify the region containing the mutation.

2. PCR Amplification: The DNA template, which contains the gene of interest, is used in a PCR reaction with the mutagenic primers. The primers bind to the flanking regions of the target sequence, and during amplification, the mutation is incorporated into the product.

3. DpnI Digestion: After PCR, the reaction mixture is treated with the restriction enzyme DpnI, which specifically digests the parental (methylated) DNA, leaving the mutated (unmethylated) DNA intact.

4. Transformation and Screening: The PCR product is then transformed into bacterial cells, where it is replicated. Bacterial colonies are screened for the presence of the mutation using techniques such as restriction digestion, sequencing, or PCR.

Significance of Site-Directed Mutagenesis:

• Studying Gene Function: This method allows researchers to study the functional effects of specific mutations in a gene.
• Protein Engineering: It is widely used to create mutant proteins for studying protein structure, function, and interactions.
• Therapeutic Applications: Site-directed mutagenesis is also used in the development of therapeutic proteins, such as creating more stable or active enzyme variants for medical applications.

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5. Explain Agrobacterium-mediated gene transfer in plant cells.

Agrobacterium-mediated gene transfer is a technique widely used in plant biotechnology to introduce foreign DNA into plant cells. This process utilizes Agrobacterium tumefaciens, a bacterium that naturally infects plants and transfers a segment of its DNA (T-DNA) into the plant genome.

Process of Agrobacterium-mediated Gene Transfer:

1. Preparation of Agrobacterium: Agrobacterium strains that contain a plasmid (such as pTi or pBIN) carrying the gene of interest are grown in culture. The plasmid also contains the T-DNA region, which is responsible for transferring the foreign gene into the plant genome.

2. Co-cultivation with Plant Cells: The plant cells, often from cultured tissues like leaf discs or stem segments, are exposed to Agrobacterium. The bacterium infects the plant cells and transfers the T-DNA region into the plant cell's nucleus, where it integrates into the plant's genome.

3. Selection of Transformed Cells: After co-cultivation, the plant cells are cultured in selective media containing an antibiotic or herbicide, which kills non-transformed cells. Only the transformed cells, which have successfully integrated the T-DNA, will survive.

4. Regeneration of Transgenic Plants: The surviving transformed cells are induced to regenerate into whole plants through tissue culture techniques. These transgenic plants contain the foreign gene, which can be expressed and studied.

Significance of Agrobacterium-mediated Gene Transfer:

• Creation of Transgenic Plants: This method is the most widely used approach for creating genetically modified plants,

allowing the introduction of traits like pest resistance, herbicide tolerance, and improved nutritional content.

• Crop Improvement: It has been used to create genetically modified crops that improve yield, resistance to diseases, and nutritional value.
• Research Applications: Agrobacterium-mediated gene transfer is also essential in plant functional genomics, helping researchers study gene function and regulation in plants.