(BT05275) GENETIC ENGINEERING

Unit I: Gene Regulation and Expression in Prokaryotes
Lactose, Arabinose and Tryptophan operons, Repressors and activator, Sigma
switch in Bacillus subtilis.
Unit II: Gene Regulation in Eukaryotic system
Gene regulation in Eukaryotic system, Repetitive DNA, Gene rearrangement,
Promoters, enhancer elements, gene amplification.
Unit III: Plasmids, Transposons / Vectors for Gene Transfers
Plasmids: Definition, types, Identification, classification and purifications and transfer
of Plasmids. Host restriction in transfer.
Transposable elements: Definition, detection of transposition in bacteria, types of
bacterial transposons, mechanisms of transposition and excision, applications of
transposons. Retrotransposons.
UNIT IV: DNA Technology
Purification of genomic DNA from living cells, Manipulation of purified DNA;
construction of prototype vector (pBR 322), different types of cloning vectors (plasmid
– pUC 19, ? phage, cosmid, M13). Enzymes involved in genetic engineering; cloning
strategies, Introduction of DNA into living cells. Methods of Gene transfer, Restriction
mapping.
UNIT – V Expression and Detection of clones
Detection of clones and its expression: Expression of cloned genes in yeast & E.
coli. Blot analysis - Southern, Northern & Western blot; dot and slot blot.
Immunological techniques. DNA methylation, DNA hybridization. Genomic and cDNA
library construction and application. DNA sequencing.
Unit VI: PCR and its application
Principles, designing of primers, PCR methodology, RT - PCR, multiplex PCR,
identification of PCR product, application of PCR technology.
Unit VII: Molecular markers
Molecular markers: RFLP, RAPD, AFLP, 16s r-ANA typing, gene chip and micro
array; applications in disease profile
Unit VIII: Applications of r-DNA Technology
Gene cloning in medicine (Insulin, Blood clotting factor VIII) High level expression
of proteins in different host systems (E. coli, yeast, Insect, mammalian cells)
Limitation and advantages and novel technologies generation of transgenic animals.
Introduction to Gene therapy (Ex vivo & In vivo), case study of ADA as an example.
Advantages and limitations of Gene therapy.
TEXT BOOKS:
1. Old RW, Primrose SB, principles of Gene manipulation, An introduction to
Genetic engineering, Blackwell Scientific Publications, 1993
2. T.A. Brown, Gene Cloning.
REFERENCES:
1. Ansubel FM., Brent A, Kingston AE, Moore DO, Current protocols in Molecular
Biology, Greene Publishing Associates, NY, 1988.
2. Berger SL, Kimmer AR, Methods in Enzymology, Vol 152, Academic Press,
1987.
3. Molecular Cell Biology – Gerald Carp.