Molecular Biology - Semester 2
Chapter 1: RNA Processing and moelcular techniques
b) Describe RNA splicing. [5]
Q. Explain phage display system. Give example
- The phage display system is a technique used in molecular biology and biotechnology to study and manipulate proteins or peptides. It involves the presentation of these molecules on the surface of bacteriophages, which are viruses that infect bacteria.
1)Gather a collection of viruses called bacteriophages. Each virus in the library carries a unique protein or peptide on its surface.
2) Insert the genetic material that codes for the displayed proteins or peptides into the phage's DNA, to create it's library.
3)Introduce the phage library to a specific molecule or protein you're interested in. The goal is to find phages that can bind to that target.
4)Remove the phages that didn't bind to the target. This step gets rid of the phages that don't interact with the target.
5) Keep the phages that successfully bind to the target. This helps enrich the population with phages that have specific binding properties.
6 ) Collect the phages that are bound to the target. These phages carry the genetic information for the protein or peptide that binds to the target.
7) Increase the number of collected phages by infecting bacteria with them. This allows for the production of a large amount of phages.
8) Repeat steps 3 to 7 several times, known as selection cycles. Each cycle helps to improve the binding properties of the phages.
9) Study the selected phages to identify the proteins or peptides responsible for binding to the target. This can involve examining the DNA or characterizing the proteins.
In developing an antibody that can specifically bind to a protein found on the surface of cancer cells. They can use the phage display system to generate a library of antibodies displayed on the surface of phages.
Q. Enlist various methods for designing probes, and explain any 2 of them.
-Methods for designing probes are:
1) PCR Primers: Polymerase Chain Reaction (PCR) primers.
2) FISH Probes: Fluorescence in situ Hybridization (FISH) probes .
3) Molecular Beacons
4) Padlock Probes
Two methods for probe design that will be explained in more detail are PCR Primers and FISH Probes:
PCR Primers:
1) PCR primers are short DNA sequences designed to specifically bind to the target DNA region of interest during the polymerase chain reaction (PCR).
2) The Primers should have similar melting temperatures to promote efficient binding to the target sequence.
3) The GC content of the primers should be optimized to ensure specific binding and stable primer-target interactions.
4) Primers should be designed to avoid self-complementarity or formation of secondary structures that may hinder PCR amplification.
5) Primers should be designed to specifically bind to the target sequence without significant off-target binding.
FISH Probes:
1) Fluorescence in situ Hybridization (FISH) probes are used to visualize specific DNA or RNA sequences in cells or tissues.
2) Determine the target sequence of interest that you want to detect using FISH.
3) Design DNA or RNA probes that are complementary to the target sequence.
4) Probes can be directly labeled with fluorescent dyes during the synthesis or post-synthesis using enzymatic or chemical labeling methods.
5) Optimize the conditions for hybridization, including the hybridization temperature, buffer composition, and washing steps, to ensure specific binding of the probes to the target sequences.
2) DNA Sequencing: Probes are used in DNA sequencing techniques to identify and determine the sequence of specific regions within a DNA molecule.
3) Diagnostic Testing: Probes are employed in diagnostic tests to detect the presence of specific DNA or RNA sequences associated with diseases or genetic disorders.
4) Fluorescence In Situ Hybridization (FISH): Probes labeled with fluorescent molecules can be used to visualize and map specific DNA sequences within cells or tissues.
5) DNA Cloning and Screening: Probes are used to identify and select specific DNA fragments of interest during the process of DNA cloning.
a) Comment on use of immunoassay. [7]
Q.Explain RNA interference.
-
- RNA interference (RNAi) is a natural process that controls gene activity in our cells.
- It uses small RNA molecules called siRNAs or miRNAs to bind to specific mRNA molecules.
- mRNA carries instructions for making proteins, but RNAi can stop the production of certain proteins by breaking down the mRNA.
- In RNAi, siRNAs or miRNAs are introduced into cells, either naturally or through experiments.
- These small RNAs team up with a protein complex called RISC.
- The RISC complex recognizes and attaches to complementary sequences on target mRNA.
- Once attached, RISC cuts and gets rid of the mRNA, preventing it from making a protein.
- By reducing specific mRNA, RNAi can lower the expression of corresponding genes.
- RNAi helps cells defend against viruses and maintain the stability of our genes.
c) Give applications of knock out mice.
- Knockout mice are genetically engineered mice in which a specific gene has been deactivated or "knocked out."
1) Gene Function Studies: Knockout mice are extensively used to study the function of specific genes.2) Disease Modeling: Knockout mice are often used to create models for various human diseases.
3) Drug Development and Testing: Knockout mice can be used to evaluate the efficacy and safety of potential drugs.
4) Toxicology Studies: Knockout mice are also employed in toxicology research to assess the impact of certain environmental factors or chemicals.
e) What is spliceosome?
-The spliceosome is like a molecular machine found in our cells. Its job is to edit the genetic instructions that are used to make proteins.
When a gene is turned into a protein, it first makes a rough draft called pre-messenger RNA (pre-mRNA). This rough draft has some extra parts in it called introns that are not needed to make the protein. The spliceosome's job is to remove these extra parts and stick the important parts called exons together.a) Justify : 'RNA interference technique can be used in gene silencing:[7]
- RNA interference (RNAi) is a powerful technique used in gene silencing, and there are several justifications for this statement:
1) Specificity: RNAi allows for highly specific gene silencing. It targets and silences the expression of specific genes without affecting the expression of other genes in the organism.
2) Versatility: RNAi can be used to silence the expression of virtually any gene, regardless of its function or location.
3) Efficiency: RNAi is a highly efficient method for gene silencing.
4) Accessibility: RNAi can be used in a wide range of organisms, from simple model organisms like fruit flies and nematodes to more complex organisms like mice and even human cells.
Chapter 2: Tools for Genetic engineering
a) Write a note on restriction endonucleases and their applications in
molecular biology. [7]
a) Comment on use of T4 DNA polymerase.
Q. Explain steps involved in CDNA construction.
- 1)The first step is to isolate RNA from the cells or tissues of interest.
2)The isolated RNA is then used as a template to synthesize cDNA. Reverse transcription is carried out using reverse transcriptase enzyme, which converts RNA into cDNA.
3)After the synthesis of the first cDNA strand, the RNA template is degraded using an enzyme called RNase H. Then, a second strand of cDNA is synthesized using DNA polymerase and dNTPs (deoxynucleotide triphosphates).
4) The double-stranded cDNA is purified to remove excess primers, enzymes, and other impurities.
5) The purified cDNA can be cloned into a suitable vector, such as a plasmid. The resulting recombinant DNA is then transformed into host cells, such as bacteria, for amplification and propagation.
6)The transformed cells are selected based on the presence of the recombinant vector. Antibiotic resistance markers or reporter genes within the vector are often used for selection.
7) Once the cDNA clones are identified, the inserts can be sequenced to determine the nucleotide sequence.
1) Total RNA is isolated from the cells or tissue of interest using a suitable RNA extraction method.
2)The isolated total RNA undergoes a step called mRNA enrichment or purification. This is done to specifically isolate the messenger RNA (mRNA) molecules from the total RNA pool.
3) The enriched mRNA is then subjected to reverse transcription. Oligo(dT) primers containing a poly(T) sequence are used to prime the synthesis of complementary DNA (cDNA) from the mRNA template.The first strand is synthesized.
4)After the synthesis of the first cDNA strand, the RNA template is degraded using an enzyme like RNase H. Then, a second strand of cDNA is synthesized.
5) The double-stranded cDNA is then treated with DNA ligase to add specific adapters or linkers at the ends of the cDNA fragments.
6) The purified cDNA is fragmented into smaller pieces using restriction enzymes. This fragmentation step helps generate cDNA fragments of manageable sizes for library construction.
7)The size-selected cDNA fragments are ligated into a suitable vector, such as a plasmid or a viral vector. The vector contains appropriate features for cloning, replication, and expression of the cDNA fragments.
8) Library Amplification and Storage: The transformed host cells are grown on agar plates containing selective media, allowing the amplification of the cDNA library.
d) Explain construction of Ti based vector.
- 1) Select a suitable Ti plasmid from Agrobacterium tumefaciens strains.
2) Remove or inactivate the tumor-inducing genes present in the Ti plasmid.
3) Design and obtain the desired genes or DNA fragments to be inserted into the vector.4) Perform recombinant DNA techniques, such as restriction enzyme digestion and DNA ligation, to insert the desired genes into the Ti-based vector.
4) Include selectable marker genes in the vector to enable the identification and selection of successfully transformed plant cells.
5) Incorporate promoters, enhancers, and other regulatory elements into the vector to control the expression of the inserted genes in plant cells.
6) Ensure the vector retains the necessary transfer functions, including the Vir genes, which facilitate the transfer of the Ti-based vector DNA into the plant genome.
7) Transform Agrobacterium tumefaciens with the constructed Ti-based vector using techniques like electroporation or heat shock.
8) Infect plant tissues with the modified Agrobacterium to allow the transfer and integration of the Ti-based vector into the plant genome.
9) Utilize plant tissue culture and regeneration techniques to obtain genetically modified plants carrying the desired traits.
| siRNA (Small Interfering RNA) | miRNA (MicroRNA) | |
|---|---|---|
| Origin | Comes from outside sources or created in the lab | Produced naturally inside our cells |
| Length | Short, about 20-25 building blocks long | Also short, about 21-25 building blocks long |
| Targeting | Silences specific mRNA molecules | Regulates the activity of multiple mRNA molecules |
| Binding | Binds perfectly to mRNA | Binds partially to mRNA |
| Function | Used in experiments to turn off specific genes | Controls gene expression during development and maintains cell functions |
| Examples | siRNA-based therapies, experimental gene silencing | let-7, miR-21, miR-155 |
b) Explain about the vectors derived from pichia and its applications.[5]
-
Here is an explanation of Pichia-derived vectors and their applications:
1) Pichia-Derived Vectors: Vectors derived from Pichia are specifically designed to work with Pichia pastoris cells for protein expression.Pichia pastoris is a type of yeast that is commonly used for protein expression.
2) These vectors typically contain genetic elements that allow for the efficient insertion and expression of foreign genes in Pichia cells.
3) Pichia-derived vectors often include strong promoters, such as the alcohol oxidase 1 (AOX1) promoter, which can drive high levels of gene expression in response to methanol induction.
4) Pichia-derived vectors have the ability to secrete recombinant proteins into the culture medium, making it easier to harvest and purify the proteins.
Applications
1) Biopharmaceutical Production: Pichia-derived vectors are extensively used for the production of biopharmaceuticals, including therapeutic proteins, vaccines, and antibodies.
2) Enzyme Production: Pichia-derived vectors are employed for the production of enzymes used in various industries, such as food and beverage, biofuel, and pharmaceutical manufacturing.
3) Protein Engineering and Functional Studies: Pichia-derived vectors are valuable tools for protein engineering and functional studies.
4) Protein-Protein Interactions: Pichia-derived vectors are used in protein-protein interaction studies.
b) What are expression vectors? Explain use of expression vectors with
suitable examples. [5]
- Expression vectors are DNA molecules commonly used in genetic engineering to express specific genes in host organisms.
1) Expression vectors are used to introduce and express foreign genes or DNA fragments in host cells, allowing the production of specific proteins of interest.
2)Expression vectors typically consist of several essential elements, including a promoter region, a coding sequence (gene of interest), a termination sequence, and a selectable marker.
3) Promoter region: The promoter is a DNA sequence that initiates gene transcription.
4) Coding sequence: This is the portion of DNA that encodes the protein of interest.
5) Termination sequence: The termination sequence signals the end of transcription and ensures proper processing of the RNA molecule.
6) Selectable marker: Expression vectors often contain a selectable marker gene, which confers a trait that allows the host cells containing the vector to be selectively grown or identified. For example, antibiotic resistance genes are commonly used as selectable markers.
7) Transformation: Expression vectors are introduced into host cells through a process called transformation.
8) Example: In biotechnology, an expression vector can be used to produce a therapeutic protein, such as insulin. The human insulin gene is inserted into an expression vector, which is then introduced into bacterial cells. The transformed bacteria can produce insulin protein, which can be harvested and used for medical purposes.
Q. Give principle and application of southern blotting.
-
Southern blotting is a technique used to detect and analyze specific DNA sequences in a sample.
1) Principle: Southern blotting is a technique used to detect and analyze specific DNA sequences in a sample.
2) DNA Separation: The first step involves the separation of DNA molecules using gel electrophoresis, which sorts them based on size.
3)Transfer to Membrane: After separation, the DNA fragments are transferred from the gel to a solid membrane, typically made of nitrocellulose or nylon.
4) Denaturation: The DNA on the membrane is then denatured to make it single-stranded, which allows for hybridization with a complementary DNA probe.
5) DNA Probe: A DNA probe is a labeled fragment of DNA that can bind specifically to the target sequence of interest.
6) Hybridization: The denatured DNA on the membrane is exposed to the DNA probe, and hybridization occurs when the probe binds to its complementary sequence.
7) Washing: The membrane is washed to remove any unbound or nonspecifically bound probe molecules.
8) Detection: The bound probe is then detected using methods such as autoradiography, fluorescence, or chemiluminescence.
9) Analysis: The resulting signal on the membrane can be visualized and quantified, allowing the identification and characterization of the specific DNA sequence in the original sample.
10) Applications: Southern blotting has various applications, including gene mapping, detection of genetic diseases and mutations, DNA fingerprinting, and studying gene expression patterns.
Q. Explain Northern blotting technique.
- 1) Northern blotting is a laboratory technique used to study gene expression by detecting and analyzing RNA molecules.
2) It is called "northern" blotting because it is a variation of the more well-known Southern blotting technique used for DNA analysis.3) The first step in northern blotting involves isolating RNA from cells or tissues of interest.
4) The isolated RNA is then separated based on size using a gel electrophoresis technique. Smaller RNA molecules move faster through the gel than larger ones.
5) After electrophoresis, the RNA molecules in the gel are transferred (blotted) onto a solid support, usually a nylon membrane.
6) The transferred RNA on the membrane is then chemically fixed, which immobilizes the RNA and preserves its spatial arrangement.
7) Next, the membrane is exposed to a labeled probe, which is a small piece of DNA or RNA that is complementary to the RNA of interest.
8) The probe hybridizes with the target RNA on the membrane, forming a stable double-stranded molecule.
9) The presence of the labeled probe is detected using techniques like autoradiography (exposing the membrane to X-ray film) or fluorescent detection.
10) By analyzing the pattern and intensity of the labeled RNA bands on the membrane, researchers can determine the size and abundance of specific RNA molecules, providing insights into gene expression levels.
Chapter 3: Genome Project
b) Write a note on - genome project of drosophila.
Q4) a) What are the salient features of human genome project. [7]
Q. What are salient features of e coli genome project.
b) Write applications of genome project.
-
Applications of the Genome Project:
1) Disease Research and Treatment: The Genome Project has contributed to our understanding of genetic factors underlying various diseases, leading to improved diagnostics, targeted therapies, and drug development.
2) Precision Medicine: Genomic information obtained from the Genome Project has paved the way for personalized or precision medicine.
3) Genetic Counseling and Screening: It provides insights into inherited genetic conditions, enabling individuals and families to make informed decisions about family planning, reproductive choices, and early intervention strategies.
4) Pharmacogenomics: The Genome Project has contributed to the field of pharmacogenomics, which examines how genetic variations influence an individual's response to medications.
5) Agricultural and Livestock Improvement: The Genome Project has had implications for agriculture and livestock by aiding in the understanding of crop and animal genomes.
f) What is gene annotation?
- Gene annotation is the process of identifying and describing the important parts of a gene within a genome. It involves finding where genes start and end, as well as determining their functions and characteristics.
This information helps scientists understand what proteins the genes can produce and how they might be involved in different biological processes.It helps identify potential protein products, predict gene functions based on similarities to known genes, and uncover regulatory elements that control gene expression.
Gene annotation is crucial for studying gene activity, analyzing genetic variations, and interpreting genomic data in various fields of research, including medicine and biology.
Chapter 4: Molecular diagnostics and applications
c) Comment on : RNA signatures of antibiotic resistance.
b) Explain the use of micro RNA in cancer diagnosis. [5]
- 1) Biomarkers for Cancer: MiRNAs exhibit altered expression patterns in cancer cells compared to normal cells.These differential expression profiles make miRNAs potential biomarkers for cancer detection and diagnosis.
2) Non-Invasive Testing: One of the major advantages of using miRNAs for cancer diagnosis is that they can be detected in easily accessible body fluids such as blood, urine, and saliva. This makes it convenient for patients and allows for the monitoring of cancer progression or treatment response over time.
3) Diagnostic Signatures: By analyzing the expression levels of multiple miRNAs simultaneously, researchers have identified specific miRNA signatures associated with different cancer types. These signatures can distinguish cancer patients.
4) Early Detection: MiRNAs can be detected at early stages of cancer development, even before clinical symptoms appear.
5) Monitoring Disease Progression: Changes in miRNA expression profiles can reflect the progression of cancer or the recurrence of the disease.
6) Research and Drug Development: MiRNAs have become targets for therapeutic interventions in cancer.
7) Personalized Medicine: MiRNA analysis in cancer diagnosis contributes to the paradigm of personalized medicine.
b) As a part of under graduate project, a student was attempting to construct
a restriction map of the plasmid pVC 23 using restriction enzymes Eco
RI and Bam HI, after carrying out both single and double enzyme
digest reactions, following fragments were obtained. [5]
Enzyme(s) Fragment length obtained
Eco RI ................................... 20 kb
Bam HI ................................ 11 kb, 6 kb, 3 kb
Eco RI + Bam HI ................ 8 kb, 6 kb, 3 kb(2)
From the information construct a restriction map of pVC 23.
Q7) Attempt the following: [Any 2] [12]
a) Give protocol for preparing gene library.
Comments
Post a Comment