BO4016 Gene Manipulation Technology Syllabus:

BO4016 Gene Manipulation Technology Syllabus – Anna University PG Syllabus Regulation 2021

OBJECTIVE

 This subject will give conceptual knowledge in the Cloning & Expression of genes, Construction of DNA libraries & Sequencing; PCR & mutagenesis; Gene transfer & Gene therapy to students.
 To gain knowledge on Gene targeting and silencing.
 To understanding the practical techniques and challenges of gene cloning.
 To understand the principles of gene transfer.
 To acquire knowledge on construction of DNA libraries.

UNIT I CLONING AND EXPRESSIONOFGENES

Overview of Restriction and Modification system. Overview of Gene Cloning; 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, Plasmids, PAC, BAC and YAC. Expression vector – Characteristics, RNA probe synthesis, High level expression of proteins, Protein solubilization, purification and export. Application of Gene Cloning – DNA analysis in research and Biotechnology.

UNIT II CONSTRUCTION OF DNALIBRARIES

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. Differences between gDNA and cDNA library. Screening strategies – Hybridization, PCR, Immuno screening, Southwestern and North-Western. Functional cloning – Functional complementation and gain of function. Difference cloning: Differential screening, Subtracted DNA library, differential display by PCR. Overview on microarray and its applications, Microarrays – Principles ofDNA microarray technology, steps and techniques involved, types of DNA arrays, applications, advantages and disadvantages.

UNIT III DNASEQUENCING

DNA sequencing – Importance, methodology, Chemical & Enzymatic methods, Pyrosequencing, How to sequence a genome? , Short gun sequencing and clone contig approach, Automated sequence, Genome sequencing methods – top down approach, bottom up approach.

UNIT IV PCRANDMUTAGENESIS

PCR – Principle and applications. Different types of PCR – Hot start PCR, Touchdown PCR, Multiplex PCR, Inverse PCR, Nested PCR, AFLP-PCR, Allele specific PCR, Assembly PCR, Asymmetric PCR, LATE-PCR, Colony PCR, in-situ PCR, Long P CR. Real-time PCR SYBR Green assay, Taqman Probes, Molecular beacons. Mutagenesis and chimeric protein engineering by PCR, RACE, Kuntel’s method of mutagenesis. Overview of types of PCR and its importance, PCR – Designing the oligonucleotide primers, studying of PCR products – Gel electrophoresis. Issues concerned with error rate of Taq polymerase. Real time PCR – Outline and concepts involved.

UNIT V GENE TRANSFER AND GENE THERAPY

Introduction to foreign genes into animal cells – Importance of DNA, Micro injection, Retroviral vectors, Trasnsfection of Embryonic stem cells, recombination. Transgenic plants – Importance Ti Plasmid, Cointegrate and Binary vectors. Overview of Gene therapy. Gene therapy – Introduction and types. Methods of Gene therapy, target sites, Gene targeting and silencing, Transgenic plants and its applications.

TOTAL : 45 PERIODS

OUTCOME

By the end of the course, the student should be able to
 detail the basic steps of gene cloning and the role of enzymes and vectors responsible for gene manipulation, transformation and genetic engineering.
 apply concept of genetic engineering techniques in basic and applied experimental biology.
 possess proficiency in designing and conducting experiments involving genetic manipulation.
 demonstrate the skills on gene manipulation, gene expression, etc which prepares them for further studies in the area of genetic engineering.
 illustrate technical know-how on versatile techniques in recombinant DNA technology.
 describe the genome editing, sequencing, and methods for gene therapy.

REFERENCES

1. Desmond Nicholl, “An Introduction to Genetic Engineering”, Cambridge University Press2002.
2. Lemonie, N. R. and Cooper, D.N. Gene Therapy, BIOS,1996.
3. Primrose S.B., Twyman R.H., and Old R.W. “Principles of Gene Manipulation”. 6th Edition., Blackwell Science, 2001
4. Winnacker E.L. “From Genes to Clones: Introduction to Gene Technology”. Panima,2003
5. Brown, T. A. (2010). Gene cloning and DNA analysis (6th ed.). Wiley Blackwell.