Syllabus | RECOMBINANT DNA TECHNOLOGY AND BIOTECHNOLOGY | Third Year | B.Sc Microbiology

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:

  1. Brown TA. (2006). Gene Cloning and DNA Analysis. 5th edition. Blackwell Publishing, Oxford, U.K
  2. Primrose SB and Twyman RM. (2006). Principles of Gene Manipulation and Genomics, 7th edition. Blackwell Publishing, Oxford, U.K.
  3. Sambrook J, Fritsch EF and Maniatis T. (2001). Molecular Cloning-A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press.
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