BY4101 Advanced Molecular Biology and Genetic Engineering Syllabus:

BY4101 Advanced Molecular Biology and Genetic Engineering Syllabus – Anna University PG Syllabus Regulation 2021

COURSE OBJECTIVES

 To understand the gene cloning methods and the tools and techniques involved in gene
 cloning and genome analysis and genomics.
 To explain the heterologous expression of cloned genes in different hosts, production of
 recombinant proteins and PCR techniques.
 To understand comparative genomics and proteomics.
 To provide extensive knowledge about Gene Regulation and Genome Editing in Genetic engineering applications.
 To explain the principles involved the creation of trangenic animals and plants.

UNIT I CLONING AND EXPRESSION OF GENES

Overview of Restriction and Modification system. Cloning vehicles: Plasmids – Host range, Copy number control, Compatibility. λ phage – Insertional and replacement vectors, in vitro packaging. Single strand DNA vector – M13 Phage. Cosmids, Phasmids, PAC, BAC and YAC. Expression vector – Characteristics, RNA probe synthesis, High level expression of proteins, Protein solubilization, purification and export.

UNIT II CONSTRUCTION OF DNA LIBRARIES

DNA library – types and importance. cDNA library: Conventional cloning strategies – Oligod T priming, self-priming and its limitations. Full length cDNA cloning – Capture method and Oligo capping. Strategies for gDNA library construction – Chromosome walking. gDNA and cDNA library. Screening strategy. Hybridization, PCR, Immuno-screening, South-Western and North Western. Functional cloning – Functional complementation and gain of function. Difference cloning: Differential screening, Subtracted DNA library, differential display by PCR. Microarrays – Applications of microarrays.

UNIT III PCR, MUTAGENESIS AND DNA SEQUENCING

Polymerase Chain Reaction (PCR): Principle and applications. Different types of PCR (Hot start, touchdown, multiplex, inverse, nested, AFLP, allele-specific, assembly, Asymmetric, LATE, Colony, in situ, long). Real-time PCR, FRET, SYBR Green assay, Taqman probes, Molecular beacons. Mutagenesis and chimeric protein engineering by PCR, RACE, Kunkels’ method of mutagenesis, Phage display and screening methodologies. DNA sequencing. Chemical and Enzymatic methods, Pyrosequencing, Automated sequencing, Genome sequencing methods – top -down approach, bottom- up approach, Next generation gene sequencing.

UNIT IV POST TRANSCRIPTIONAL GENE REGULATION AND GENOME EDITING

Role of siRNA, miRNA in gene regulation: siRNA- PTGS, Quelling, origin, components of gene silencing mechanisms, TGS, PTGS, Applications. miRNA- Identification, biogenesis, mechanism of action. Difference between siRNA and miRNA, effect of small RNAs on chromosomal DNA. Genome Editing tools: Genome editing- introduction, zinc finger nucleases, TALENS, CRISPRCas9 systems, applications.

UNIT V GENETIC ENGINEERING

Introduction of foreign genes into animal cells – Importance, DNA Microinjection, Retroviral vectors, Transfection of Embryonic stem cells, recombination. Transgenic plants –Ti Plasmid, Co integrate and Binary vectors, transgenic plants for disease resistance, abiotic stress resistance, enhanced nutritional value, Viral vectors, Engineering siRNA mediated gene knock downs (shRNA).

TOTAL : 45 PERIODS

COURSE OUTCOMES

After completion of the course the students will be able to
CO1 Gain knowledge in gene cloning methods and the tools and techniques involved in gene cloning and genome analysis and genomics.
CO2 Understand the heterologous expression of cloned genes in different hosts, production of recombinant proteins and PCR techniques.
CO3 Gain extensive knowledge in comparative genomics and proteomics.
CO4 Gain extensive knowledge about Gene Regulation and Genome Editing in Genetic engineering applications.
CO5 Understand the principles involved the creation of trangenic animals and plants.

REFERENCES:

1. Primrose S.B., Twyman R.H., and Old R.W. “Principles of Gene Manipulation.” 7th Edition. Blackwell Science/Oxford, 2006
2. Winnacker E.L. “Frome Genes to Clones: Introduction to Gene Technology.” Panima, 2003.
3. Glick B.R. and Pasternak J.J. “Molecular Biotechnology: Principles and Applications of Recombinant DNA, 3rd Edition. ASM Press, 2003.
4. Lemonie, N.R. and Cooper, D.N. “Gene Therapy.” BIOS Scientific, 1996
5. Nelle W. and Hammann C. “Small RNAs: Analysis and Regulatory Functions (Nucleic
Acids and Molecular Biology)”, 2nd Edition. Springer Verlag Berlin and Heidelberg GmbH & Co. K, 2007
6. Krishnarao Appasani. “Genome Editing and Engineering” Cambridge University press 2018.
7. Raghavachari Nalini, Garcia-Reyero Natàlia. “Gene expression analysis: Methods and protocols” 1st Edition, Humana Press, 2018.