SUBJECT: RECOMBINANT DNA TECHNOLOGY AND BIOTECHNOLOGY
| S.no | TOPIC | DOMAIN | HOURS |
| 1 | Introduction to biotechnology and basic DNA cloning
Milestones in genetic engineering and biotechnology. Simple cloning of DNA fragments, Vectors: Definition and properties.. Transformation of DNA by chemical method and electroporation.
|
Must know | 10 |
| E. coli expression vectors-lac, tac and T7 promoter based vectors. Yeast expression vectors – pET yeast vectors, YIp, YEp and YCp vectors | Desirable to know | 5 | |
| Baculovirus based vectors. Ti based vectors (Binary and Cointegrated vectors) and cloning using linkers and adaptors. | Nice to know | 5 | |
| 2 | Tools of recombinant DNA technology
Hosts and enzymes: Agrobacterium-mediated delivery E. coli strains; Yeast (Saccharomyces cerevisiae, Pichia pastoris); Fungi (Penicillium, Aspergillus); Mammalian cell lines – names and genotypes. Restriction modification systems: Types I, II and III. Mode of action, nomenclature. Application of Type II restriction enzymes in genetic engineering. DNA modifying enzymes and their applications Vectors: Cloning Vectors- Definition and Properties. Plasmid vectors-pBR and pUC series, Bacteriophage lambda and M13 based vectors. Cosmids. Shuttle vectors. BACs, YACs, MACs. |
Must know | 14 |
| Terminal deoxynucleotidyl transferase, kinases and phosphatases, DNA ligases and DNA polymerases, reverse transcriptases, bacteriophage RNA polymerases, exonuclease III, BAL31, mung bean nuclease, S1 nuclease. | Desirable to know | 4 | |
| Mammalian Expression Vectors- SV40, Vaccinia, Retroviral promoter based vectors. | Nice to know | 2 | |
| 3 | Gene delivery and amplification of nucleic acids: Microinjection, biolistic method (gene gun), liposome and viral-mediated delivery, Agrobacterium-mediated delivery Polymerase chain reaction – enzymes used, primer design.
Analytical methods and DNA typing: Agarose gel electrophoresis, Southern – and Northern – blotting techniques, dot blot and colony hybridizations. Chromosome walking and jumping. DNA fingerprinting by RFLP and RAPD. SDS-PAGE and Western blotting. Phage display.
|
Must know | 13 |
| Cloning PCR products. RT-PCR and principles of real time PCR. Ligation chain reaction. | Desirable to know | 4 | |
| Gel retardation assays. DNA footprinting by DNase I, DNA microarray analysis. | Nice to know | 3 | |
| 4 | Construction of genomic libraries: Genomic and cDNA libraries: Preparation and uses. Screening of libraries by colony hybridization and colony PCR.
DNA sequencing and product of DNA technology: Maxam-Gilbert’s and Sanger’s method. Automated sequencing. Human genome sequencing project. Bt transgenics-rice, cotton, brinjal |
Must know | 14 |
| Human protein replacements-insulin, hGH and Factor VIII. | Desirable to know | 3 | |
| Human therapies – tPA, interferon, antisense molecules. | Nice to know | 3 | |
| 5 | PRACTICAL
Digestion of DNA using restriction enzymes and analysis by agarose gel electrophoresis. Ligation of DNA fragments. |
Must know | 40 |
| Demonstration of PCR. | Desirable to know | 10 | |
| Interpretation of sequencing gel electropherograms. | Nice to know | 10 |
Suggested readings:
- Brown TA. (2006). Gene Cloning and DNA Analysis. 5th edition. Blackwell Publishing, Oxford, U.K
- Primrose SB and Twyman RM. (2006). Principles of Gene Manipulation and Genomics, 7th edition. Blackwell Publishing, Oxford, U.K.
- Sambrook J, Fritsch EF and Maniatis T. (2001). Molecular Cloning-A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press.