BO4004 Techniques in Molecular Biology and Genetic Engineering Syllabus:
BO4004 Techniques in Molecular Biology and Genetic Engineering Syllabus – Anna University PG Syllabus Regulation 2021
COURSE OBJECTIVES:
The course aims to
> Enlightenkey molecular biology and genetic engineering techniques
> Applythe latest techniques in current biological research as well as in biotechnology industries
UNIT I VECTOR SYSTEMS
Overview of tools in recombinant DNA technology. Artificial chromosomes – YACs and BACs. Principles for maximizing gene expression – expression vectors, pMal, GST, pET-based vectors. Protein purification – His-tag, GST-tag and MBP-tag. Intein-based vectors; Inclusion bodies; methodologies to reduce formation of inclusion bodies; mammalian expression and replicating vectors; Baculovirus and Pichia vectors system, plant based vectors, Ti and Ri plasmids as vectors, yeast vectors and shuttle vectors.
UNIT II ASSAY TECHNIQUES IN MOLECULAR BIOLOGY
Nuclease protection assays, Nuclease S1 mapping, Reporter assays – Mono and dual reporter assays, Electrophoretic mobility shift assay (EMSA) / Gel shift assay, Run-off transcription assay, Phage display, Ribosome display, Gene silencing – siRNAs and Morpholinos.
UNIT III HIGH-THROUGHPUT DNA SEQUENCING
Preparation of Next Generation Sequencing (NGS) libraries: Fragmentation versus tagmentation, end repair, clonal amplification – Bridge PCR and emulsion PCR. Basics and steps involved in NGS platforms: Illumina/Solexa, Roche 454, Ion-torrent and Pacific biosciences. Current status of Oxford nanopore sequencing. Principles of Mate pair sequencing, ChIP-seq, RIP/CLIP-Seq, Methyl seq – Restriction enzyme, enrichment and bisulfite treatment strategies.
UNIT IV GENE EXPRESSION ANALYSIS
Overview of gene expression and its significance. Hybridization methods: Southern and Northern. PCR methods: Reverse transcriptase PCR, End point Vs Real time PCR, Relative quantitation, Absolute quantification – Standard curve method and digital PCR. Endogenous/loading controls. High throughput analysis: Multiplex PCR, Microarray, Serial analysis of gene expression (SAGE) and Small Amplified RNA-SAGE (SAR-SAGE), Total analysis of gene expression (TOGA), Gene calling, RNA-seq and Ribosome profiling.
UNIT V GENOME EDITING TECHNOLOGIES
Basics and applications of genome editing methods – Zinc-finger nuclease (ZFN), Transcription activator-like effector nucleases (TALEN), Mega nucleases, CRISPR-Cas systems – Types and applications, Homing endonucleases, Transposons and Cre/lox P systems. Gene delivery systems – Physicochemical methods and viral vectors.
TOTAL: 45 PERIODS
COURSE OUTCOMES:
At the end of the course, the students will be able to
CO1. detail the basic steps of gene cloning and the role of enzymes and vectors responsible for gene manipulation, transformation and genetic engineering.
CO2. apply concept of genetic engineering techniques in basic and applied experimental biology.
CO3. possess proficiency in designing and conducting experiments involving genetic manipulation.
CO4. demonstrate the skills on gene manipulation, gene expression, etc which prepares them for further studies in the area of genetic engineering.
CO5. Illustrate technical know-how on versatile techniques in recombinant DNA technology.
CO6. describe the genome editing and sequencing and methods for gene therapy.
REFERENCES:
1. Steven R. Head, Phillip Ordoukhanian, Daniel R. Salomon. “Next Generation Sequencing: Methods and protocols” 1st Edition, Humana Press, 2018.
2. Krishnarao Appasani. “Genome Editing and Engineering” Cambridge University press 2018.
3. Raghavachari Nalini, Garcia-Reyero Natàlia. “Gene expression analysis: Methods and protocols” 1st Edition, Humana Press, 2018.
4. Primrose SB and Twyman RB. “Principles of Gene manipulation and Genomics”. 7th Edition, Wiley-Blackwell, 2006.
5. Green MR and Sambrook J. “Molecular Cloning: A Laboratory Manual”. 4th Edition, CSHL press, 2012.