SRI KRISHNADEVARAYA UNIVERSITY:: ANANTAPUR M.Sc. BIOTECHNOLOGY (
SRI KRISHNADEVARAYA UNIVERSITY:: ANANTAPUR
M.Sc. BIOTECHNOLOGY (SEMESTER SYSTEM)
(Effective from 2005 -2006)
COURSE PATTERN AND SCHEME OF EXAMINATION
Title and Subject of the paper Work Examination
week Duration Max. Marks
Paper I Cell Biology and Microbiology 4 3 100
Paper II Biomolecules 4 3 100
Paper III Biochemical and Biophysical
Techniques 4 3 100
Paper IV Biodiversity and Genetics 4 3 100
Practical I Cell Biology, Microbiology and 6 6 100 *
Practical II Analytical Techniques and 6 6 100 *
Theory 4 4 100
Paper V Enzymology and Intermediary 4 4 100
Metabolism 4 4 100
Paper VI Immunology and 4 4 100
Paper VII Molecular Biology
Paper VIII Genetic Engineering
6 6 100 *
6 6 100 *
Practical III Enzymology and
Practical IV Molecular Biology and Genetic
Title and Subject of the paper Work Examination
Paper IX Plant Biotechnology 4 3 100
Paper X Animal Biotechnology 4 3 100
Paper XI Microbial Biotechnology 4 3
Paper XII Biostatistics, Bioinformatics and 100
Biosafety 4 3
Practicals 100 *
Practical V Plant and Animal Biotechnology
Practical VI Microbial Biotechnology, 6 6
Biostatistics and Bioinformatics
3 months 150
* Inclusive of 10 marks for Record and 10 marks for Viva-voce
SRI KRISHNADEVARAYA UNIVERSITY:: ANANTAPUR
M.Sc. BIOTECHNOLOGY (SEMESTER SYSTEM)
Paper I: CELL BIOLOGY AND MICROBIOLOGY
Microscopy - Principles of compound, phase contrast, fluorescence, scanning and transmission
electron microscopy. Cytophotometry and flow cytometry. Cell structure and organization in
bacteria, higher plant and animal cells. Comparison of prokaryotic and eukaryotic cells.
Fixation, smearing and staining of cell and microscopic examination of cells. Cell components
and organelles – Cell wall, lysosomes, ribosome, golgi complex, peroxisomes, glyoxysomes,
mitochondria, plastids, endoplasmic reticulum, vacuoles, centrioles and cytoskeleton.
Chromosomes – Nucleosome, Packing of DNA into chromatin. Euchromatin and
heterochromatin. Chromatin folding into chromosome. Special types of chromosomes –
polytene, lamp brush and accessory chromosomes. Genomes of mitochondria and plastids.
Cell division and Cell cycle - Events of cell and cell division in prokaryotes and eukaryotes.
Cell cycle regulation. Mitosis and meiosis, and apoptosis. Structure of biomembranes –
Chemical composition of animal and plant cell membranes. Assembly of membrane
components. Micelle, liposome and biomembranes. Fluid mosaic model of membranes.
Membrane transport - Donnan membrane equilibrium, passive, mediated and active transport.
Mechanism of active transport (Primary and Secondary – Na+, K+, Glucose transport).
Membrane channels and pumps. Receptors – Chemical nature. Signal transduction – Concept of
second messengers (cAMP, Ca2+) and their role.
Historical development of microbiology – spontaneous generation, germ theory of disease.
General characteristics of protozoa, fungi, algae, cyanobacteria, rickettsiae, mycoplasma,
spirochetes and archaebacteria. Outlines of classification of bacteria as per Bergey’s Manual of
systematic Bacteriology. Nomenclature rules – International code. Chemotaxonomy of
bacteria. Nucleic acid based classification of bacteria. Ultrastructure of bacteria – An account of
variant (capsule, flagellum, pilus, plasmids, endospore) and invariant (cell wall, cell membrane,
mesosomes, nucleoid, ribosome), components of bacterial cell. Morphology and the use in
modern biology of E.coli, Bacillus sp., Rhizobium sp., Agrobacterium sp.
Nutritional requirements in bacteria – Photoautotrophs, chemoautotrophs, photoheterotrophs,
organotrophs, methylotrophs, mixotrophs, auxotrophs. Methods in Microbiology – Different
methods of sterilization. Chemotherapy/Antibiotics – Antimicrobial agents, sulfa drugs;
antibiotics; penicillin and cephalosporin. Broad spectrum antibiotics. Antibiotics from
prokaryotes. Antifungal antibiotics. Mode of action (Penicillin, tetracycline), Resistance to
antibiotics. bacterial and fungal toxins. Preparation of culture media. Isolation of pure culture.
Culture collection and maintenance of cultures.
Microbial growth – Growth curves, measurement of growth and growth yields. Synchronous
growth, continuous culture by chemostats and turbidostats. Growth as affected by environmental
factors like temperature, acidity, alkalinity, water availability and oxygen.
Microbial diseases – Diseases, reservoirs, epidemiological terminologies. Tuberculosis.
Sexually-transmitted diseases including AIDS. Diseases transmitted by animals (rabies, plague),
insects and ticks (rickettsias, lyme disease, malaria), food and water–borne diseases. Pathogenic
fungi. Introduction to virology - Nomenclature and classification of viruses, morphological and
biochemical properties, ultra structure of TMV, Caulimovirus, M13 ans SV40. Lysogeny and
lytic cycle in bacteriophage. Biology of sub-viral particles – prions, viroids, satellite viruses,
defective interfering particles.
1. Genes VI. B. Lewin..
2. The Making of a Fly. Lawrence. 1992. Backwell & Scientific Co. Ltd., Oxford.
3. Annual Review of Cell Biology, Krumlaui. 1992. Volume 8.
4. Cell and Molecular Biology, De Roberties & De Robertis
5. The Cell, Vols. I-V, Brachl & Mirsky.
6. Cell and Molecular Biology, Dupre.
7. The Cell, Tawcott.
8. Molecular Biology of Cell, Alberts et al
9. Cell Biology, David E. Sadava
1. Pelczar, M.J., Chan, E.C.S. and Krieg, N.R. 1986.Microbiology, V edition,
McGraw Hill Book Co.
2. Stanier, R.Y., Adelberg, F.A. and Ingraham, J.L. 1984. General Microbiology, IV
Edition, McMillan Press.
3. Prescott, L..M., Harley, J.P. and Klein, D.A. 1990. Microbiology, WCB Publishers.
4. Rosenbeng, E. and Chohan, I.R. 1983. Microbial Biology, HS International Editions.
5. Powar, C.B. and Daginwala, H.F. 1989. General Microbiology, Vol. II and I.
Himalaya Publishing House.
6. Volk, W.A. and Wheeler, M.F. 1980. Basic Microbiology, J.B. Lippincott Company.
7. Buffaloe, H.D. and Ferguson, D.V. 1981.Microbiology. Houghton Mifflin Company.
8. Freifelder, D. 1987. Microbial Genetics. Jones and Bartlett Publ.
9. Hayes, W. 1988. The Genetics of bacteria and their viruses. John Wiley and Sons.
10. Jacob, F. and Wollman, C.D. 1961. Sexuality and Genetics of bacteria. Academic Press.
Paper II: BIOMOLECULES
Chemical foundation of biology – pH, pK, acids, bases, chemical bonding, properties of water.
Carbohydrates – Classification and properties of carbohydrates, mono (glucose, galactose,
fructose) di (lactose, maltose, sucrose), poly (starch, glycogen, cellulose). Chemical and
enzymatic methods for structural elucidation of starch and mucopolysaccharides. Lipids –
Classification. Structure and biological functions of fatty acids, triacylglycerols, phospholipids,
steroids. Physico-chemical properties and analysis of fats and oils. Structure and functions of
prostaglandins, leukotrienes, thromboxanes.
Amino acids – Classification. Structure and physicochemical properties. Peptides - Peptide
bonds. Naturally-occurring peptides (glutathione, bradykinin, kallikrien, tyrocidin). Peptide
synthesis by solid-phase technique. Proteins – Classification. Isolation and purification of
protein, criteria of homogeneity. Primary structure of proteins and its sequence determination.
Secondary (Ramachandran plot), tertiary and quaternary structural features of proteins, Primary
structure and protein folding (Alfensen’s experiment on ribonuclease). Forces responsible for
protein stability. Structural organization of globular (myoglobin, hemoglobin), fibrous proteins
(collagen, keratins, silk fibroin). Denaturation and renaturation of proteins, chaotropic agents.
Structure and functions of glycoproteins and lipoproteins.
Nucleic Acids – Structure of purines, pyrimidine, nucleosides, and nucleotides. Structure,
properties and functions of nucleic acids (DNA, RNA). Different forms of DNA and RNA.
Three dimensional structure of RNA. Isolation of nucleic acids, Denaturation and renaturation
of nucleic acids, cot curves, chemical synthesis of DNA. The law of DNA constancy and C-
1. Textbook of Biochemistry. 1968 by West and Todd (MacMillan).
0. Principles of Biochemistry. 1993 by A.L. Lehninger, Nelson and Cox. (C.B.S., India).
0. Principles of Biochemistry General Aspects. 1983 by Smith et al., (McGraw Hill).
0. Biochemistry (2nd edition) by Donald Voet and Judith Voet.
0. Biochemistry (4th edition) by L. Stryer (Free man).
0. Textbook of Biochemistry with Clinical Correlation (4th edition) by Thomas M. Devlin.
0. Biochemistry by Zubay.
0. Nucleic acid Biochemistry and Molecular Biology by Main Waring et al., (Blackwell).
0. Biochemistry, 2nd edition by Albert L. Lehninger. 1978. Kalyani Publishers, New Delhi.
0. Biochemical calculations, Irwin H.Segel, John Wiley and sons Inc
0. Organic Chemistry, DJ Cram and GS Hammond
Paper III: BIOCHEMICAL AND BIOPHYSICAL TECHNIQUES
pH Determination – Measurement of pH, biochemical buffers, selection of biochemical buffer,
oxygen electrode and biosensors. Homogenization – Cell disruption methods: mechanical and
non-mechanical methods including French press, sonication, freeze-thaw techniques, use of
liquid nitrogen in cell disruption. Centrifugation – Basic principles of sedimentation,
sedimentation coefficient and Svedberg unit, types of centrifuges and rotors. Preparative
ultracentrifugation - differential centrifugation, density gradient, analytical ultracentrifugation
and applications in determination of molecular weight, purity and detection of conformational
changes in macromolecules.
Separation methods - General principles and definitions. Methods based on polarity – Partition
chromatography, adsorption chromatography, gas liquid chromatography, reverse phase liquid
chromatography. Methods based on size - Gel filtration, principle, methodology and
applications. Dialysis, ultra filtration, lyophilization. Methods based on affinity – Affinity
chromatography, principle, methodology, applications (purification of proteins, nucleic acids).
High performance liquid chromatography – Principle, instrumentation, practical procedure,
analysis of results and applications: FPLC, hydrophobic interaction chromatography.
Ion-exchange chromatography – Principle, ion exchangers, methodology, pH and salt gradients
for elution of proteins, amino acids and nucleotides. Amino acid analyzer. Electrophoresis –
General principles and definitions.
PAGE – Native-PAGE, SDS-PAGE, Isoelectricfocussing, 2D electrophoresis, identification of
novel proteins in 2D gels, capillary electrophoresis.
Agarose gel electrophoresis - Preparation, separation and determination of molecular size of
DNA, denaturing agarose gel electrophoresis and their applications, recovery of DNA from
agarose gels. Pulse-field gel electrophoresis – principle, methodology and applications in
separation of large DNA fragment. Methods to study macromolecular interactions – Mobility
shift gel electrophoresis, DNA foot printing, use of fusion proteins to study the protein
interactions – GFP, FRET, GST and coimmunoprecipation.
Spectroscopy – Principles, laws of light absorption, Instrumentation and applications of UV-
visible spectrophotometer, fluorescence spectroscopy, NMR, ESR, IR and Raman spectroscopy,
Mass spectroscopy – MALDI, TOF. Radioisotope Techniques – Types of isotopes, radioactive
decay. Detection and measurement of radioactivity – GM counter, scintillation counter,
autoradiography and Cerenkov counting. Preparation of label compounds – pulse chase studies
and tracer techniques, isotopes used in biology, safety methods in handling radioisotopes.
Optical activity of biomolecules - CD and ORD – principles and applications. X-Ray diffraction
– Basic principles, applications in the determination molecular structure of protein and nucleic
1. Separation methods in biochemistry by S.J. Morris and P. Morris (Pitman)
2. The tools of Biochemistry by Terrance G. Cooper (Wiley)
3. Biochemical research technique (A practical introduction by Ed. John M. Wriggles worth
4. Analytical biochemistry by David J. Holmes and Hazel peck
5. A Biologist's guide to principles and techniques of practical biochemistry, 2nd edition Ed. by
BL. Williams and K. Wilson (Edward Arnold)
6. Biophysical chemistry D. Freifelder, W.H. Freeman
7 Experimental techin. Ex ques in biochemistry by Drewer Pesec, AJ. And As worth, R.B.
8. Principles of Physical Biochemistry by K.E. Vanholdem W.C. Johnson, P.S. Ho, (Prentice
9. Manometer and biochemical techniques by W.W Umbrit and R.H. Burris (Burgens
10. The determination of Molecular Structure by P.J. Wheatly (Oxford Clarenders press) 1968
11. Molecular Biophysics – R.B. Setlow and E.C. Pollard (Addison Wiley publishing co.
12. Biophysics – Edited by W. Hoppe, W. Lehman Hi. Maskal and H. Ziegler (Springer verlag,
13. Practical biochemistry: Principles & Techniques – Edited by Wilson & Walker, Cambridge
Univ. Press, New York) 1986.
14. Biological Spectroscopy by Iain D. Campbell and Raymond A. Dwek, the
Benjamin/Cumming Pub. Co., California, London.
15. Biophysical Chemistry by C.R. Cantor and P.R. Schimmel, W.H. Freeman & Co., NY.
16. Introduction to Biophysical Methods for Protein and Nucleic Acid Research, Ed. J.A. Glasel
and M.P. Deutscher, (Academics Press), 1995.
Paper IV: BIODIVERSITY AND GENETICS
Biodiversity - an outline, definition, levels of diversity, animals, plants and microbial diversity,
terrestrial and aquatic diversity of genes (gene diversity). Species diversity – Measurement,
concept of species, richness, abundance, turnover relationships, global distribution of species,
centers of species diversity, mega diversity centers, hot spot analysis. Biodiversity versus
biotechnology, biodiversity vs. bioproductivity, bioprospects. Conservation of biodiversity –
principle and rationale, ex situ and in situ. Conservation - global conservation programs, social
movements. Sustainable management of biodiversity, national and international policies.
Classical genetics - Outlines of Mendilian genetics. Linkage, crossing over and chromosome
mapping in eukaryotes. Numerical and structural variants of chromosome – aneuploidy,
polyploidy, deletion, duplication, translocation and inversion. Mutations – molecular basis of
mutation and mutagenesis, and Ames test. Extra-chromosomal genetics. Genomes of
mitochondria, choroloplast, plasmids and transposable elements (transposans, Ty, Copia, P
elements). Molecular mechanism of replicative transposition.
Identification of nucleic acids as genetic material. One gene – one polypeptide concept. Modern
concept of a gene. Benezer’s experiments on r II locus of T4 phage. Cis-trans test. Concept of
cistrons, recons and mutons. Gene clusters, single copy genes and unique sequences.
Repetitive sequences in genic and non-genic DNA. Split genes, overlapping genes and
oncogenes. Plasmids – types, incompatibility, amplification, curing.
Methods of gene transfer in bacteria: Transformation – Development of competence in
Hemophilus. In vitro transformation – Cold CaCl2 technique, electroporation. Conjugation -
Hfr strains, transfer of F, Hfr chromosomes. Transduction - generalized and specialized.
Abortive transduction, cotransduction and sexduction. Use of recombination frequency,
interrupted mating and transduction for genentic mapping.
10. K.V.Krishnamurthy. 2003. An advanced text book on Biodiversity, Principles and Practice.
Oxford & IBH Publishers. New Delhi.
10. Wilson, E.O and F M Peters (Eds) Biodiversity. National Academy Press Washington.
3. Pushpangadan, P., K.Ravi and V. Santhosh (eds) Conservation and Economic evaluation
of Biodiversity. Oxford & IBH Publishers. New Delhi.
1. Stickburgher - Genetics
2. Murray (Ed.) 1991. Gene transfer and expression protocols. Methods in Molecular Biology.
Humana Press, Chifton, N.J., pp 439.
3. Lewin, B., Genes VI, Oxford University Press, pp. 857.
4. Suzuki – Principles of Genetic Analysis.
5. Freifelder, D. 1994. Microbial Genetics, Springer.
6. Latchman, D. 1994. Gene regulation (3nd edn.), Sciential Publ.
Practical I: CELL BIOLOGY, MICROBIOLOGY AND BIOMOLECULES
0. Microscopic examination of different cell types in plants and animals.
0. Study of mitochondria by vital staining
0. Study of haemolysis suing RBC for cell permeability
0. Analysis of mitotic stages
0. Isolation of chloroplast and demonstration of Hill reaction
0. Preparation of haemin crystals
0. Preparation of blood smear and cell count
0. Study of Cheek epithelium
0. Assay of Na+- K+ - ATPase.
0. Donnan membrane equilbrium.
11. Subcellular fractionation and marker enzymes from rat liver
0. Preparation of liquid and solid media for growth of microorganisms.
0. Isolation and maintenance of microorganisms by streaking and serial dilution plating
4. Isolation of pure cultures of microorganisms from soil and water
4. Slants and stab culture. Storage of microorganisms
5. Growth; Microbial growth curve; Measurement of bacterial population by turbidometry and
serial dilution methods. Effect of temperature, pH and carbon and nitrogen sources on
6. Microscopic examination of bacteria, yeast and molds and study of organisms by Gram stain,
acid fast stain and staining for spores.
7. Assay of antibiotics and demonstration of antibiotic resistance.
8. Analysis for water potability and determination of MPN
9. Bacterial transformation
10. Biochemical characterization of selected microbes
0. Reactions of amino acids and sugars
0. Isolation and estimation of glycogen from liver
0. Isolation and estimation of Cholesterol from brain
0. Preparation of lecithin
0. Estimation of proteins by Biuret, modified Lowry and Bradford method.
0. Estimation of amino acids by ninhydrin method.
0. Estimation of glucose by glucose oxidase method
0. Titration curve of an amino acid and calculation of pK and pI values.
Practical II: ANALYTICAL TECHNIQUES AND GENETICS
1. Preparation of buffers and measurement of pH
2. Separation and identification of amino acids by paper chromatography.
3. Separation and identification of sugars by TLC.
4. Separation and identification of lipids by TLC.
5 Separation of amino acids by Ion-exchange chromatography.
6. Separation of proteins by Gel filtration
7. Absorption spectra of amino acids, Proteins and nucleic acids
8. Verification of Beer’s law.
9. Determination of molar extinction coefficient of p-nitrophenol.
10. Measurement of inversion of sucrose by polarimetry.
12. HPLC demonstration
1. Bacterial transformation
2. Bacterial conjugation
3. Isolation of auxotrophic mutants
4. Isolation of streptomycin-resistant mutants.
5. Genetics problems based on theory syllabus.
SRI KRISHNADEVARAYA UNIVERSITY: ANANTAPUR
M.Sc. BIOTECHNOLOGY (SEMESTER SYSTEM)
Paper V: ENZYMOLOGY AND INTERMEDIARY METABOLISM
IUB nomenclature and classification of enzymes. Nature of enzymes, Active site, coenzymes,
assay of enzyme, units of enzyme activity. Kinetics of enzyme catalysed reaction - Michaelis-
Menten equation Determination of Vmax, Km, Kcat, and their significance. Effect of pH,
temperature, concentration of substrate and enzyme concentration, organic solvents on enzyme
catalysis and structural consequences. Activators and inhibitors. Enzyme inhibition – reversible
and irreversible. Competitive, non-competitive and feed-back enzyme inhibition, enzyme
poisoning. Mechanisms of enzyme action – lysozyme, chymotrypsin. Enzyme regulation –
Allosteric modification, covalent modification, zymogens activation. Isoenzymes (LDH),
Multienzyme, complexes, Multifunctional enzymes, Modern concepts of evolution of catalysis –
Concept of free energy. Thermodynamic principles in biology. Uptake and utilization of
nutrients, High energy compounds, Biological energy transducers, Glycolysis, Citric acid cycle,
Glyoxylate cycle, HMP shunt, Gluconeogenesis. Components and organization of mitochondrial
electron transport system (experimental approach). Mechanism of oxidative phosphorylation.
Metabolic pathways involved in production of ethanol, acetic acid, glutamic acid, citric acid and
Photosynthetic pigments and photosynthesis in bacteria and higher plants. Chloroplast as an
energy transducing organelle. Cyclic and non-cyclic photophosphorylation. Mechanism of
photophosphorylation. Pathways of CO2 fixation by C3, C4 and CAM pathways.
Photorespiration. Fatty acid oxidation (ß-oxidation). Biosynthesis of fatty acids, triglycerides,
Coordinated synthesis of purine and pyrimidine nucleotides. Synthesis of essential and
nonessential amino acids, formation of ammonia and urea. Events triggering secondary
metabolite production. Synthesis of vitamins (vit B12 and riboflavin, vitamin A), antibiotics
(Penicillin, streptomycin), tannins, alkaloids, saponins and toxins (aflotoxins, endotoxins- BT
0. Principles of Biochemistry: White. A., Handler, P. and Smith.
0. Biochemistry: Lehninger, A.L.
0. Biochemistry: David E. Metzler
0. Biochemistry: Lubert stryer.
0. Review of Physiological Chemistry: Harold A. Harper
0. Outlines of Biochemistry: Conn and Stummf.
0. Text of Biochemistry: West and Todd.
0. Metabolic Pathways: Greenberg
0. Mitochondria: Munn.
0. Biochemistry: 2nd Edition
0. Biochemistry - G. Zubay (1988).
0. Mandelstam & Maxuilam – Microbial Metabolism
0. Microbiology – Pelczar.
Paper VI: IMMUNOLOGY AND IMMUNOTECHNOLOGY
Phylogeny of immune system. Types of immunity - innate and acquired. Cells of the immune
system - B cells, T cells, NK cells, phagocytes, inflammatory cells, antigen presenting cells,
organs - primary, secondary and tertiary lymphoid organs. Antigens - nature, types, factors
influencing antigenecity, haptens, adjuvants and superantigens. Antibodies - structure, types,
classes and functions. Antibody diversity – theories of antibody diversity, mechanism of
diversification, allelic exclusion.
T cell receptor - structure and diversity. MHC - Types, structure, distribution, self restriction, T
and B cell activation. Maturation of lymphocytes – positive and negative selection, process of
maturation. Antigen processing and presentation – cytosolic and endosomal pathways, T and
NK cell - mediated lysis of cells, ADCC. Complement system - components, cascades, MAC,
outcomes. Cytokines – classification, properties and role as immunomodulators.
Hypersensitivity – classification, mediators, mechanism, consequences of hypersensitive
reactions. Autoimmunity - concept of tolerance, types of autoimmune disorders, basis and
therapy for autoimmune disorders. Transplantation – transplantation antigens, mechanism of
graft rejection, graft versus host reaction, immunosupressors. Tumor immunity - tumors of
immune system, immune responses against tumors. Immunodeficiency - primary and secondary
immunodeficiency, combined immunodeficiency, complement deficiency, AIDS.
Antigen–antibody interactions - principle, lattice hypothesis. Precipitation reaction - radial
immunodiffusion, ouchterlony technique, immunoelectrophoresis, counter current and rocket
electrophoresis. Agglutination reactions - bacterial and hemeagglutination, passive
agglutionation, agglutination inhibition assay. RIA and ELISA – principle, methodology and
application. Immunofluorescence, FACS, immunoblotting. Hybridoma technology – poly
clonals, monoclonals, selection, HAT Medium, production of monoclonal antibodies and
applications. Vaccines - concept of immunization, routes of vaccination. Types of vaccines -
whole organism (attenuated and inactivated) and component vaccines (synthetic peptides, DNA
vaccines, recombinant vaccines, subunit vaccines).
1. Essentials of Immunology - Ian & Roitt - Blackwell Scinentific Publications.
2. Fundamentals of immunology - William C. Boyed (Wiley Toppan).
3. Introduction to Immunology - John W. Kinball.
4. Fundamentals of Immunology - Otto S. View and others.
5. Immunology - D.M. Wier.
5. Immunology - Jains Kubay, (2001) Second Edition, W H Frecman & Com. New York.
7. Cellular and Molecular Immunology 3rd ed. Abul K. Abbas Andrew
K. Lichtman Jordan S. Pober
Paper VII: MOLECULAR BIOLOGY
DNA replication – Modes of replication. Messelson–Stahl experiment and Cairns studies.
Initiation, elongation and termination of DNA replication in bacteria, eukaryotes and viruses.
DNA polymerases, klenow fragment, DNA ligases, topoisomerases. DNA methylation.
Regulation of ColE1 plasmid replication. DNA Damage and Repair - DNA damage by UV,
alkylating agents, cross linkers. Mechanism of repair – photo reactivation, excision repair,
recombination repair, recA in repair, SOS. Adaptive response and its regulation.
Transcription (RNA biosynthesis) – RNA polymerases in prokaryotes and eukaryotes.
Identification and characterization of promoters. Initiation, elongation and termination of RNA
synthesis. Monocistronic and polycistronic mRNAs. Post-transcriptional modifications, RNA
replicase in RNA viruses. Polynucleotide phosphorylase reaction, reverse transcription.
Inhibitors of RNA synthesis.
Translation (protein biosynthesis) – Elucidation of the genetic code, methods employed to
decipher genetic code. Activation of amino acids, initiation, elongation and termination of
polypeptide chain synthesis. Post-translational modification of proteins. Protein channeling.
Inhibitors of protein synthesis.
Regulation of gene expression – Levels of regulation. The operon concept. Enzyme induction
and repression. Regulatory genes, structural genes and repressors. Negative and positive
regulation. Regulation of lac, ara, trp operons. Regulation of gene expression in lambda and nif
operon. Regulation of gene expression in eukaryotes – gal operon in yeast. Hormones and
environmental factors affecting gene expression, antisense RNA and its role in regulation of
eukaryotic genes. Molecular biology of cancer, oncogenes, chemical carcinogenesis.
1 Molecular Biology of the Gene (4th Edn) JD Watson, NH Hopkins, JW Roberts,
JA Steitz and AM Weiner, The Bjnjamin/Cummings Publ, Co. Inc, California.
2 Molecular Cell Biology (2nd Edn) J. Darnell, H.Lodish and D. Baltimore,
Scientific American Books, Inc. USA 1994
3. Molecular Cloning: A Laboratory manual, J. Sambrook, E.Ffrisch and T.
Maniatis, Old Spring Harbor Laboratory Press New York, 2000
4. Introduction to Practical Molecular Biology, P.D. Dabre, John Wiley & Sons Ltd,
5. Molecular Biology, TA Brown (Ed) Bios Scientific Publishers Ltd.,Oxford, 1991
Paper VIII: GENETIC ENGINEERING
Genetic engineering - Introduction and outlines of gene cloning. DNA cutting and joining -
Enzymatic cleavage of DNA. Restriction and modification enzymes – Classification,
nomenclature and importance of restriction endonucleases. Restriction mapping, DNA ligases,
polynucleotide kinase, alkaline phosphotases, S1 nuclease, terminal transferase, Bal 31 nuclease.
Polymerase chain reaction - principle, types (RT-PCR, nested and inverse PCR) and
applications. Sequencing methods – Sanger’s, and Maxam-Gilbert’s method. Automated
Cloning vectors - Characteristics of a vector. Natural plasmids used as vectors – advantages
and disadvantages. Artificial plasmids and their importance as cloning vectors. Vectors used for
cloning in E.coli. (plasmids, bacteriophage derivatives, cosmids), BACs, yeast (YAC, shuttle
vectors), higher plants (Ti plasmid, caulimovirus) and animal cells (constructs of SV 40 and
retroviruses). Characteristics of expression vectors. Generation of DNA fragments containing a
gene (shot-gun method, southern analysis, and cDNA synthesis). Joining of DNA fragments to
vector molecules, cohesive termini ligation and blunt end ligation - linkers, adaptors and
homopolymer tails. Construction of DNA libraries – genomic and cDNA libraries. Screening
of recombinants for a positive clone.
Introduction of Recombinant DNA molecules into appropriate hosts – Competent cells
preparation, electroporation, microinjection and particle bombardment method, and selection of
transformants. Agrobacterium - mediated transformation of plant cells – Identification of
transformed cells and micropropagation of transformed cell into callus, and regeneration of
Transfection - Salient features and its significance in transforming animal cells. In vitro
packaging of recombinant cosmids. Introduction of recombinant cosmids into E.coli cells.
Detection of clones with desired genes - Genetic methods, nucleic acid hybridization methods,
colony hybridization, immunochemical methods – ELISA and its significance in identification
of clones with desired genes.
Expression of cloned genes - Construction of expression vectors: Vectors having inducible lac,
taq promoters. Use of IPTG and its role in induction of a cloned gene. Expression vector
constructed using T7 promoter. Expression of proteins with His tag and its significance in
simultaneous expression and purification of recombinant proteins.
Cloning strategies – Strategies for cloning and over expression of proteins with therapeutic
values. Somatostatin, insulin, interferon and growth hormones. Cloning and expression of
proteins with immunogenic properties – vaccines. Production technologies – synthesis of
plantibodies. Problems associated with expression of cloned genes – inclusion bodies,
solubilisation and reconstruction of expressed proteins.
DNA Finger Printing - RAPD, RFLP and AFLP analysis. Markers linked to drug and disease
resistant genes. Application of RFLP in forensic science, pedigree analysis, biodiversity,
genetic counseling and germ plasm maintenance. Antisense technology and its application. Use
of knockout organisms and gene replacement. Use of transposon tagging.
1. Molecular Cloning: A Laboratory manual, J. Sambrook, E.Ffrisch and T. Maniatis, Old
Spring Harbor Laboratory Press New York, 2000
2. DNA Cloning : a Practical Approach, DM Glover and BD Hames, IRL Press
3. Molecular and Cellular methods in Biology and Medicine. PB Kaufman,W.Wu.D Kim and
LJ Cseke, CRC
4. Methods in EnzymologyVol. 152, Guide to Molecular Cloning Techniques, SL Berger and
AP Kimmel, Academic Press, Inc San Diego, 1998
5. Methods in Enzymology Vol. 185, Gene Expression Technology, D V Goeddel,
Academic Press, Inc. San Diego, 1990
6. DNA Science. A first Course in Recombinant Technology, DA Mickloss and GA Freyer,
Cold Spring Harbor Laboratory Press, New York 1990
7. Molecular Biotechnology (2nd Edn) SB Primrose, Blackwell Scietific Pub. Oxford, 1994
8. Milestones in Biotechnology. Classic papers on Genetic Engineering. JA Davies and WS
Reznikoff, Butterworth-Heinemann, Boston, 1992
9. Route Maps in Gene Technology, MR Walker and R Rapley, Biackwell Science
Ltd, Oxford 1997
10. Molecular Biotechnology – Glick
11. Concepts of Biotechnology D. Balasubramanian
12. Principles of Gene Manipulation by Old and Primrose, Blackwell
Practical III: ENZYMOLOGY AND IMMUNOTECHNOLOGY
0. Assay of trypsin
0. Assay of L-amylase from saliva
0. Assay of LDH from serum
0. Assay of acid and alkaline phosphatase
0. A preparation of urease crystals from horsegram seeds and assay
0. Purification of an enzyme and effect of time pH, temperature, substrate concentration,
enzyme concentration, inhibition on enzyme activity.
0. Molecular weight determination of enzyme by SDS - PAGE
1. a. Staining of blood smear and identification of different lukocytes.
b Determination of A,B, O and Rh blood groups in human beings.
2. Identification of primary and secondary lymphoid organs.
3. Recognition of T-cell by rosette formation.
4. Preparation of antigen.
5. Injection of antigen into experimental animals by different routes.
6. Bleeding of test animals for antiserum.
7. Electrophoretic study of normal and immune serum.
8. Preparation of immunoglobulins from serum.
9. Separation of immunoglobulins by gel electrophoresis.
10. Labelling of purified immunoglobulins with enzymes/dyes.
11. Different serological tests
a. Agglutination - Brucella system
b. Precipition test - Ring interface and agar gel double diffusion tests.
c. Immunoelectrophoresis - Counter current and Rocket.
d. Hemagglutination and Hemagglulination inhibition tests.
e. Labeled antibody tests - ELISA, Dot ELISA, FAT and Western blotting.
e. Viral infectivity neutralization test.
12. Cell - mediated immunity tests
a. Leukocyte migration inhibition test
b. Opsonic index.
Practical IV: MOLECULAR BIOLOGY AND GENETIC ENGINEERING
0. Isolation of genomic DNA from bacteria, rice and sheep liver
0. Determination of purity of the isolated DNA by UV spectrophometry
0. Quintification of DNA
. Spectroscopic method (UV absorption method)
. Colorimetric method (Diplhenylamine reagent)
4. Thermal denaturation of DNA and demonstration of hyperchromic effect.
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