Honours / Discipline Specific Elective (DSE)
►Unit 1: Introduction
Organization of E.coli and Drosophila genome.
Q. What is conjugation?
Conjugation is the direct transfer of genetic material from one bacterium to another. In conjugation, two bacteria lie close together and a connection forms between them. A plasmid or a part of the bacterial chromosome passes from one cell (the donor) to the other (the recipient). Subsequent to conjugation, crossing over takes place between homologous sequences in the transferred DNA and the chromosome of the recipient cell. In conjugation, DNA is transferred only from donor to recipient, with no reciprocal exchange of genetic material.
►Unit 2: Molecular Techniques in Gene manipulation
Recombinant DNA technology, Restriction endonucleases. Cloning Vectors & their features: plasmids, Phage vectors, Cosmids, Phagemids, BAC, YAC, and HAC. Shuttle and Expression Vectors. Construction of Genomic libraries and cDNA libraries Transformation techniques: Cloning in bacteria and detection technique of clone Agarose and Polyacrylamide Gel Electrophoresis, Southern, Northern and Western blotting, Polymerase chain reaction: Allele specific, RAPD & RT PCR, DNA Fingerprinting.
Q. Define Neo-schizomer and Isoschizomer with examples.
Restriction enzymes with the same sequence specificity and cut site are known as isoschizomers. SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other. The first enzyme discovered which recognizes a given sequence is known as the prototype; all subsequently identified enzymes that recognize that sequence are isoschizomers. Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.
Enzymes that recognize the same sequence but cleave at different points, for example SmaI (CCC/GGG) and XmaI C/CCGGG), are sometimes known as neoschizomers.
Q. What is ARMS-PCR? State its limitations
The allele-specific PCR is also called as the (amplification refractory mutation system) ARMS-PCR because of the use of two different primers for two different alleles. Here the word “refractory” is very important (Refractory= resistant to something). Two sets of primers are designed, the mutant set of the primer is refractory (resistant) to the normal PCR and the normal set of the primers are refractory to the mutant PCR reaction. That is why it is called an amplification refractory mutation system. The name ARMS-PCR is given by its actual developer C. R. Newton.
Deletion/other major duplication and chromosomal abnormalities cannot be detected, Only known SNPs are detected by ARMS-PCR, Internal control is required because of the chance of the false-negative results, It is temperature-sensitive. A minute fluctuation in the temperature leads to false-positive results, Thousands of SNPs cannot be detected in a single assay.
►Unit 3: Genetically Modified Organisms
Production of cloned and transgenic animals: Nuclear Transplantation, Retroviral Method, DNA microinjection. Applications of transgenic animals: Production of pharmaceuticals, production of donor organs, knock-out mice.
Q.Why mice is used in knock out experiment?
i)Mice are currently the laboratory animal species most closely related to humans for which the knockout technique can easily be applied. They are widely used in knockout experiments, especially those investigating genetic questions that relate to human physiology. Gene knockout in rats is much harder and has only been possible since 2003.
ii)Knocking out the activity of a gene provides valuable clues about what that gene normally does. Humans share many genes with mice. Consequently, observing the characteristics of knockout mice gives researchers information that can be used to better understand how a similar gene may cause or contribute to disease in humans.
iii)Examples of research in which knockout mice have been useful include studying and modeling different kinds of cancer, obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease. Knockout mice also offer a biological context in which drugs and other therapies can be developed and tested.
iv)Many of these mouse models are named after the gene that has been inactivated. For example, the p53 knockout mouse is named after the p53 gene, which … (For further information follow our contact section)
►Unit 4: Culture Techniques and Applications
Animal cell culture, Expressing cloned genes in mammalian cells, Molecular diagnosis of genetic diseases (Cystic fibrosis, Sickle cell anaemia, Thalassemia). Dolly &Polly cloning Genetically modified economically important animal Gene Therapy.
Q. Briefly describe the process of molecular diagnosis of Cystic fibrosis.
The diagnosis of CF requires clinical symptoms consistent with CF in at least one organ system and evidence of CFTR dysfunction usually based on an abnormal sweat chloride test or the presence of mutations in the CFTR gene.
Clinical symptoms aren’t required for infants identified through newborn screening. Other diagnostic tests that may be performed include:
(i)Immunoreactive trypsinogen (IRT) test
The immunoreactive trypsinogen (IRT) test is a standard newborn screening test that checks for abnormal levels of the protein called IRT in the blood.
A high level of IRT may be a sign of cystic fibrosis. However, further testing is required to confirm the diagnosis.
(ii)Sweat chloride test
The sweat chloride test is the most commonly used test for diagnosing cystic fibrosis. It checks for increased levels of salt in the sweat. The test is performed by using a chemical that makes the skin sweat when triggered by a weak electric current.
Sweat is collected on a pad or paper and then analyzed. A diagnosis of cystic fibrosis is made if the sweat is saltier than normal.
During a sputum test, the doctor takes a sample of mucus. The sample can confirm the presence of a lung infection. It can also show the types of germs that are present and determine which antibiotics work best to treat them.
A chest X-ray is useful in revealing swelling in the lungs due to blockages in the respiratory passageways.
A CT scan creates detailed images of the body by using a combination of X-rays taken from many different directions.
These images allows your doctor to view internal structures, such as the liver and pancreas, making it easier to assess the extent of organ damage caused by cystic fibrosis.
(vi)Pulmonary function tests (PFTs)
Pulmonary function tests (PFTs) determine whether your lungs are working properly
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