SUBJECT: RECOMBINANT DNA TECHNOLOGY AND BIOTECHNOLOGY
|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.
|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.
|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.
|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
|Human protein replacements-insulin, hGH and Factor VIII.||Desirable to know||3|
|Human therapies – tPA, interferon, antisense molecules.||Nice to know||3|
Digestion of DNA using restriction enzymes and analysis by agarose gel electrophoresis.
Ligation of DNA fragments.
|Demonstration of PCR.||Desirable to know||10|
|Interpretation of sequencing gel electropherograms.||Nice to know||10|
- 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.