KUK - LMS

Syllabus

MODULE NO / UNIT	COURSE SYLLABUS CONTENTS OF MODULE	NOTES
Course Code: MMB-301 Course Name: Recombinant DNA Technology
1	Basics of DNA cloning, and methods of DNA and protein analysis: Simple cloning and cloning using linkers and adaptors. Cloning into various kinds of vectors – plasmids, phages lambda and M13, phagemids, cosmids, P1 phage, PACs, BACs and YACs. Selection and screening of clones. Southern and Northern Blotting. Radiolabelling probes. Isolation and purification of DNA. RFLP analysis. DNA fingerprinting and its application in forensics, in disease diagnosis and in identification of strains. Western Blotting analysis. Polymerase chain reaction and construction of cDNA and genomic DNA libraries: Concept of PCR and various thermophilic enzymes used in PCR. Gradient PCR versusTouchdown PCR. Designing primers. Cloning PCR products. Long PCR, Inverse PCR,Vectorette PCR, RT-PCR, 5’ and 3’ RACE, Real Time PCR using SYBR Green, Scorpion primers and TaqMan probes, MOPAC, Multiplex PCR, Differential Display PCR, RAPD fingerprinting of micro-organisms, Ligation Chain Reaction, Overlap PCR, Rolling Circle Amplification Technology. Vectors used in the construction of cDNA versus genomic DNA libraries. Steps in the construction of cDNA versus genomic DNA libraries. Screening libraries by colony hybridization and colony PCR. Screening expression libraries. Enriching for clones in cDNA libraries by positive selection and subtractive hybridization. Identifying genes in complex genomes by direct selection of cDNA and exon trapping.
2	Genome sequencing: DNA sequencing by Sanger’s method – traditional and cycle sequencing. Physical mapping by restriction fragment fingerprinting of BAC clones and STS mapping. E-PCR. Whole genome shotgun sequencing. Clone-by-clone shotgun sequencing of genome – preparation of BAC/YAC library, selection of BACs, subclone library construction, random shotgun phase and finishing phase followed by sequence authentication. Next Generation sequencing methods.
3	Transcriptional analysis of gene expression and transcriptomics: Gene expression analysis by Northern Blotting, RT-PCR, EST analysis and the use of reporter genes. Enzymatic and bioluminescent reporters. Reporters used in protein localization and trafficking studies. Promoter analysis – deletion analysis and linker scanning analysis coupled to reporter assays, mapping transcriptional start sites by S1 nuclease mapping, primer extension studies or 5’ RACE. Transcriptome analysis by DD-PCR and EST analysis, DNA microarrays (cDNA arrays and oligo arrays), Serial Analysis of Gene Expresion (SAGE), RNA-seq. Overexpression of recombinant proteins: Overexpression and tagging of recombinant proteins in E.coli, driven by lac, T7 and Tet-regulatable promoters, Expression in B. subtilis. Overexpression systems in S.cerevisiae, P.pastoris, S.pombe and K.lactis. Baculovirus overexpression system. Mammalian cell overexpression system.
4	Analysis of protein-DNA and protein-protein interactions, protein engineering and proteome analysis: Gel retardation assay, DNA footprinting by DNase I and chemical methods, yeast one-hybrid assay, ChIP- chip, ChIP-seq. Yeast two hybrid, three-hybrid, split hybrids and reverse hybrid. Co-immunoprecipitation, pull-down, far-western. Use of GFP and its variants in FRET analysis, use of BiFC. Phage display. Insertional and deletion mutagenesis. Site directed mutagenesis by conventional and PCR-based methods. Proteome analysis by 2D gel electrophoresis coupled to mass spectrometric analysis. Principles and used of MALDI-TOF and LC-MS platforms. PMF verses MS/MS. Protein arrays and their applications. Applications of recombinant DNA technology: Human protein replacements – insulin, hGH and Factor VIII. Human therapies – TPA, interferon, antisense molecules. Vaccines – Hepatitis B, AIDS, and DNA vaccines. Creating transgenic animals and plants.