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GENETIC ENGINEERING
The objectives of this course are to teach students with various approaches to conducting genetic engineering and their applications in biological research as well as in biotechnology industries.
Genetic engineering is a technology that has been developed based on our fundamental understanding of the principles of molecular biology and this is reflected in the contents of this course.
Impact of genetic engineering in modern society; general requirements for performing a genetic engineering experiment; restriction endonucleases and methylases; DNA ligase, Klenow enzyme, T4 DNA polymerase, polynucleotide kinase, alkaline phosphatase; cohesive and blunt end ligation; linkers; adaptors; homopolymeric tailing; labelling of DNA: nick translation, random priming, radioactive and non-radioactive probes, hybridization techniques: northern, southern, south-western and far-western and colony hybridization, fluorescence in situ hybridization.
Plasmids; Bacteriophages; M13 mp vectors; PUC19 and Bluescript vectors, hagemids; Lambda vectors; Insertion and Replacement vectors; Cosmids; Artificial chromosome vectors (YACs; BACs); Principles for maximizing gene expression expression vectors; pMal; GST; pET-based vectors; Protein purification; His-tag; GST-tag; MBP-tag etc.; 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 as vectors, yeast vectors, shuttle vectors.
Principles of PCR: primer design; fidelity of thermostable enzymes; DNA polymerases; types of PCR – multiplex, nested; reverse-transcription PCR, real time PCR, touchdown PCR, hot start PCR, colony PCR, asymmetric PCR, cloning of PCR products; T-vectors; proof reading enzymes; PCR based site specific mutagenesis; PCR in molecular diagnostics; viral and bacterial detection; sequencing methods; enzymatic DNA sequencing; chemical sequencing of DNA; automated DNA sequencing; RNA sequencing; chemical synthesis of oligonucleotides; mutation detection: SSCP, DGGE, RFLP.
Insertion of foreign DNA into host cells; transformation, electroporation, transfection; construction of libraries; isolation of mRNA and total RNA; reverse transcriptase and cDNA synthesis; cDNA and genomic libraries; construction of microarrays – genomic arrays, cDNA arrays and oligo arrays; study of protein-DNA interactions: electrophoretic mobility shift assay; DNase footprinting; methyl interference assay, chromatin immunoprecipitation; protein-protein interactions using yeast two-hybrid system; phage display.
Gene silencing techniques; introduction to siRNA; siRNA technology; Micro RNA; construction of siRNA vectors; principle and application of gene silencing; gene knockouts and gene therapy; creation of transgenic plants; debate over GM crops; introduction to methods of genetic manipulation in different model systems e.g. fruit flies (Drosophila), worms (C. elegans), frogs (Xenopus), fish (zebra fish) and chick; Transgenics - gene replacement; gene targeting; creation of transgenic and knock-out mice; disease model; introduction to genome editing by CRISPR-CAS with specific emphasis on Chinese and American clinical trials.
• Brown, T. A., Gene Cloning and DNA Analysis, An Introduction. Wiley –Blackwell publication. 2010.
• Sandy B. Primrose, Richard M. Twyman. Principles of Gene Manipulation and Genomics, Blackwell Sciectific Publication. 2009.
• Brown, T. A., Genomes (3rd ed.). New York: Garland Science Pub. 2006.
• Old, R. W., Primrose, S. B., & Twyman, R. M., Principles of Gene Manipulation: an Introduction to Genetic Engineering. Oxford: Blackwell Scientific Publications. 2001.
• Green, M. R., & Sambrook, J., Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 2012.
• Selected papers from scientific journals, particularly Nature & Science.
• P.B. Kaufman, W. Wu. D. Kim and L.J; Cseke, Molecular and Cellular Methods in Biology and Medicine, CRC Press, Florida.
• M. Glover and B.D. Hames, DNA Cloning: a Practical Approach, IRL Press, Oxford.
• S.L. Berger and A.R. Kimmel, Methods in Enzymology vol. 152, Guide to Molecular Cloning Techniques, Academic Press, Inc. San Diego.
• D.V. Goeddel, Methods in Enzymology Vol 185, Gene Expression Technology, Academic Press, Inc., San Diego.
• Kingsman S.M. and Kingsman A.J., Genetic Engineering. An Introduction to gene analysis and exploitation in eukaryotes. Blackwell Scientific Publications, Oxford, 1990.
• Desmond S.T. Nicholl, An Introduction to Genetic Engineering. Edited by, Cambridge University Press. 2002.
• Sue Carson and Dominique Robertson, Manipulation and Expression of Recombinant DNA., Second edition, Academic Press. 2005.
• Primrose and Twyman, Principles of Gene Manipulation and Genomics. (7th edition). Blackwell Publishers. 2006.
