Syllabus (MICROBIOLOGY)

Course Type: MAJ-8

Semester: 6

Course Code: BMCBMAJ06T

Course Title: RECOMBINANT DNA TECHONOLOGY

(L-P-Tu): 4-2-0

Credit: 6

Practical/Theory: Combined

Course Objective: 1. To learnthe scope and importance of recombinant DNA technology. 2. To study the different molecular methods and vectors used widely used in RDT.

Learning Outcome: Upon successful completion of the course the candidate will be able to - 1. Understand the importance of genetic engineering and its application in various fields. 2. Gain knowledge ofbasic molecular tools and molecular methods used in recombinant DNA technology. 3. Understand the different gene cloning vectors and the techniques in transformation of recombinant DNA into target host organisms. 4. Gain the concept of DNA amplification, gene sequencing, blotting techniques and construction of cDNA and genomic libraries.

MAJOR COURSE 01

COURSE TITLE: RECOMBINANT DNA TECHNOLOGY (RDT)(THEORY)

TOTAL HOURS:60 CREDITS:4

Unit 1. Molecular Cloning- Tools and Strategies No. of hours: 14

Basic concepts of genetic engineering, Cloning Tools; Restriction modification systems: Types I, II and III. Mode of action, nomenclature, applications of Type II restriction enzymes in genetic engineering DNA modifying enzymes and their applications: DNA polymerases. Terminal deoxynucleotidyltransferase, kinases and phosphatases, and DNA ligases, Cloning Vectors: Definition and Properties Plasmid vectors: pBR and pUC series, Bacteriophage lambda and M13 based vectors Cosmids, BACs, YACs, Use of linkers and adaptors, Expression vectors: E. coli lac and T7 promoter-based vectors.

Unit 2. Methods in Molecular Cloning No. of hours:12

Transformation of DNA: Chemical method, Electroporation, Gene delivery: Microinjection, electroporation, biolistic method (gene gun), liposome and viral mediated delivery, Agrobacterium - mediated delivery, DNA, RNA and Protein analysis: Agarose gel electrophoresis, Southern - and Northern – blotting techniques, dot blot, DNA microarray analysis, SDS-PAGE and Western blotting.

Unit 3. DNA Amplification, DNA sequencing No. of hours: 12

PCR: Basics of PCR, RT-PCR, Real-Time PCR, Sanger’s method of DNA Sequencing: traditional and automated sequencing, primer walking and shotgun sequencing

Unit 4. Construction and Screening of Genomic and cDNA libraries No. of hours:12

Genomic and cDNA libraries: Preparation and uses, Screening of libraries: Colony hybridization and colony PCR, Chromosome walking and chromosome jumping

Unit 5. Applications of Recombinant DNA Technology No. of hours: 10

Products of recombinant DNA technology: Products of human therapeutic interest - insulin, hGH, antisense molecules. Bt transgenic - cotton, Gene therapy, recombinant vaccines, protein engineering and site directed mutagenesis

SUGGESTED READING

1. 1. Brown TA. (2010). Gene Cloning and DNA Analysis. 6th edition. Blackwell Publishing, Oxford,U.K.
   2. Clark DP and Pazdernik NJ. (2009). Biotechnology: Applying the Genetic Revolution. ElsevierAcademic Press, USA
2. Primrose SB and Twyman RM. (2006). Principles of Gene Manipulation and Genomics, 7th edition, Blackwell Publishing, Oxford, U.K.
3. Sambrook J and Russell D. (2001). Molecular Cloning-A Laboratory Manual. 3rd edition. ColdSpring Harbor Laboratory Press
4. Wiley JM, Sherwood LM and Woolverton CJ. (2008). Prescott, Harley and Klein’s Microbiology.McGraw Hill Higher Education
5. Brown TA. (2007). Genomes-3. Garland Science Publishers
6. Primrose SB and Twyman RM. (2008). Genomics: Applications in human biology. BlackwellPublishing, Oxford, U.K
7. Russell PJ. (2009). i Genetics- A Molecular Approach. 3rd Ed, Benjamin Cummings

COURSE TITLE: RECOMBINANT DNA TECHNOLOGY (RDT) (PRACTICALS)

TOTAL HOURS: 60 CREDITS:2

1. Isolation of plasmid DNA.
2. Digestion of DNA using restriction enzymes and analysis by agarose gel electrophoresis.
3. Demonstration of blue-white screening.
4. Demonstration of Southern blotting.