Genetic Engineering is concerned with deliberate manipulation of the genetic material (DNA) of a living organism by artificial methods.
This course is divided into four units:
GE 341: Genetic Engineering
DNA & Basics Of Recombinant DNA Technology
Structure of DNA: A-,B-,Z-, and triplex DNA, measurement of properties, spectrophotometric, CD, AFM, and electron microscope analysis of DNA structure.
Restriction analysis: Types of restriction enzyme, Type I, II and III, restriction modification systems, type II restriction endonucleases and properties, isoschizomers and neoschizomers, mcr/mrr genotypes, Cohesive and blunt end ligation, linkers, adaptors, homopolymeric tailing.
Labeling of DNA: Nick translation, random priming, radioactive and non-radioactive probes, use of Klenow enzyme, T4 DNA polymerase, bacterial alkaline phosphatase, polynucleotide kinase. Hybridization techniques: Northern, Southern and Colony hybridization, Fluorescence in situ hybridization Restriction maps and mapping techniques, DNA fingerprinting, chromosome walking & chromosome jumping.
DNA-Protein Interactions: Electro mobility shift assay, DNase I footprinting, methyl interference assay
Gene Cloning Vectors: Plasmids, bacteriophages, Cloning in M13 mp vectors, phagemids, Lambda vectors; insertion and replacement vectors, EMBL, λDASH, λgt10/11, λZAP etc. Cosmid vectors. Artificial chromosome vectors (YACs, BACs), Animal Virus derived vectors- SV-40, vaccinia/bacculo & retroviral vectors. Expression vectors; pMal, GST, pET-based vectors. Protein purification; His-tag, GST-tag, MBP-tag etc. Restriction proteases, intein-based vectors. Inclusion bodies, methodologies to reduce formation of inclusion bodies. Baculovirus and pichia vectors system
Insertion of Foreign DNA into Host Cells: Transformation, Transfection: Chemical and physical methods, liposomes, microinjection, macroinjection, electroporation, biolistics, somatic cell fusion, gene transfer by pronuclear microinjection, Plant transformation technology: Basis of tumor formation, hairy root, features of Ti and Ri plasmids, mechanism of DNA transfer, role of virulence genes, use of Ti and Ri as vectors. Cloning and expression in yeasts (Saccharomyces, Pichia etc.), animal and plants cells, methods of selection and screening, cDNA and genomic cloning, expression cloning, jumping and hopping libraries, southwestern and far western cloning, yeast two hybrid system, phage display, Construction of cDNA libraries in plasmids and screening methodologies, Construction of cDNA and genomic DNA libraries in lambda vector. Principles in maximizing gene expression, Site-directed mutagenesis.
PCR and Its Applications
Primer design, Fidelity of thermostable enzymes, DNA polymerases, multiplex, nested, reverse transcriptase, real time PCR, touchdown PCR, hot start PCR, colony PCR, cloning of PCR products, T-vectors, proof reading enzymes, PCR in gene recombination, deletion, addition, overlap extension, and SOEing, site specific mutagenesis, PCR in molecular diagnostics, viral and bacterial detection, PCR based mutagenesis.
Sequencing methods: Enzymatic DNA sequencing, Chemical sequencing of DNA, principle of automated DNA sequencing, RNA sequencing. Chemical Synthesis of oligonucleotides. Gene silencing techniques: Introduction to siRNA and siRNA technology, micro RNA, construction of siRNA vectors, principle and application of gene silencing. Gene knockouts and Gene Therapy: Creation of knock out mice, disease model, somatic and germ-line therapy in vivo and ex-vivo, suicide gene therapy, gene replacement, gene targeting
Other applications: Transgenics, Genome projects and their implications, application in global gene expression analysis. Applications of recombinant DNA technology in medicine, agriculture, veterinary sciences.
- Teacher: Sandip Kale Biochem