301 GENETIC ENGINEERING
Unit I
1. The recombinant DNA Technology : General concept and principle of cloning
2. Enzymes: Nucleases and restriction endonucleases- properties and types;
phosphomonoesterases; polymerase; terminal deoxynucleotidyl transferase; poly A
polymerase, Linkers, adaptors and homopolymer tailing.
3. prokaryotic host- vector system: Characteristics of E.coli as host; vectors for cloning in
E.coli (plasmid, bacteriophage- EMBL, λDASH, λgt10/11,λ ZAP etc and plasmidphage),
4. Other Prokaryotic host vector systems: BAC ,Characteristics of Gram positive and
Gram negative organism as host and suitable vectors for cloning; Shuttle Vectors
Unit II
1. Design and characteristics of expression vectors for cloning in prokaryotes and factors
that affect expression.
2. Cloning in Yeast: Properties of yeast as host for cloning and different types of vectors
designed for cloning in yeast
3. Cloning in animal system: Animal system as a model host, Methods of introduction of
foreign DNA in animal system; Vectors for cloning in animal system- SV-40, vaccinia
virus, baculovirus and retrovirus vectors ,pMal, GST, pET based vectors, Pichia based
vectors.
4. Plant transformation technology: Features of Ti and Ri plasmids, mechanism of DNA
transfer.
Unit III
1. Methods for Constructing rDNA and cloning: Inserts; vector insert ligation; infection,
transferring and cloning
2. Methods for screening and selection of recombinant clones
3. DNA Libraries: types, advantages and disadvantages of different types of libraries;
Different methods for constructing genomic and full length cDNA libraries
4. Gross anatomy of cloned insert- size, restriction mapping and location
Unit IV
1. Fine anatomy of DNA segment- General principle of chemical and enzymatic methods
of nucleotide sequence analysis and advantages of automatic gene sequencers.
2. Localization of cloned segments in genomes- molecular and chromosomal location
3. Methods for determination of copy number of a cloned gene in genome
4. Mutant construction: Introduction, deletion, insertion and point mutation
Unit V
1. Principles and applications of Blotting techniques- Southern, Northern, Western and
Eastern blotting; Polymerase Chain reaction and types (multiplex, nested, RT, real time,
touch down PCR, hot start PCR, colony PCR), Oligonucleotide
2. Principle and applications of gel mobility shift assay, DNA fingerprinting and DNA
Foot printing, restriction fragment length polymorphism, Chromosome mapping and
chromosome painting
3. Application of Recombinant DNA technology in Medicine & Industry
4. Si RNA and si RNA technology: Micro RNA Construction of si RNA vectors: Gene
silencing and its applications in agro industry.
Practical Exercises
1. Bacterial Culture and antibiotic selection media. Preparation of competent cells
2. Isolation of plasmid DNA
3. Isolation of phage DNA
4. Quantitation of nucleic acids
5. Restriction mapping of plasmid DNA
6. Cloning in plasmid/phagemid vectors
7. Preparation of helper phage and its titration
8. Preparation of single stranded DNA template
9. Gene expression in E .coli and analysis of gene product
10. Polymerase Chain Reaction
Reference Books
1. Recombinant DNA – By Watson et al
2. Principles of Gene Manipulation, Old and Primrose
3. Gene Cloning: An introduction , Brown
4. Biotechnology: Theory and Techniques (Vol I & II, 1995), Chirikjian
5. Molecular Genetics of Bacteria , Dale
6. Molecular Cloning (Vol I, II & III, 2001), Sambrook & Russell
7. Applied Molecular Genetics (1999), Miesfeld
8. Genes and Genome (1991), Singer & Berg
9. Molecular Biotechnology , Glick & Pasternak
10. Plant Molecular Biology (Vol I & II, 2002), Gilmartin & Bowler
