CONTENTS:
Introduction: what is genetic engineering; organisation of living system and flow of genetic information. Genome and gene organisation.
Working with nucleic acids: isolation of DNA and RNA, handling and quantification of nucleic acids. Restriction enzymes, ligases and modifying enzymes
Labelling of nucleic acids, Nucleic acid hybridization, DNA sequencing
Gene manipulation: host cell types, vector types; preparation of competent host, getting DNA into cell
Cloning strategies: synthesis of cDNA, cloning of cDNA into plasmid and bacteriophage vector, cDNA library, preparation of genomic DNA for cloning, genomic library
Polymerase chain reaction: methodology, tips and tricks, exotic PCR techniques (nested, inverse), mutagenesis by PCR
Selection, screening and analysis of recombinants: genetic selection an screening; nucleic acid hybridization, immunological screening for expressed genes
Gene expression: northern blotting, RNase protection assay, in situ hybridisation, real time PCR, microarray, affymetrix,
Cloning into expression vector: characteristics of expression vectors, types of tags on recombinant proteins
Recombinant protein expression, recombinant protein purification, protein-protein interactions (pull down and yeast two hybrid system); tandem affinity protein purification, immunoprecipitation
How target protein influence phenotype: insertion mutagenesis and gene trap vectors, generation of knockout and knockdown mutants, RNAi,
Commercial application of genetic engineering: recombinant proteins in pharmaceutical industry, genetically modified plants, transgenic animals
LEARNING OUTCOMES:
Understand how genes could be cloned and methods of nucleic acid cloning
Understand the procedures and methods of labeling, modifying and quantifying nucleic acids
Understand principles of gene manipulation: gene deletion, mutation and overexpression
Know how to prepare and analyze the gene and/or genome bank
Know the basic properties of the expression vectors and the basic principles of recombinant proteins expression
Understand and apply the methods for recombinant protein purification
Understand and apply the methods to study protein protein interactions
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- Predavanja koja su dostupna u PDF formatu na Web stranicama PMFa.
- 1. Molecular Biotechnology: Principles and Applications of Recombinant DNA, (2009), Ed. Bernard R. Glick, ASM Press
2. Nicholl DST, 2008: An Introduction to Genetic Engineering, 3rd Edition, Cambridge University press
3.Grossman,W. R. and Moldave, K. (1989), Recombinant DNA Methodology, Acad. Press, San Diego
4. Hackett, P. B., Fuchs,E. F., and Messing, J.W. (1988), An introduction to recombinant DNA techniques. Basic experiments in gene manipulation. The Benjamin-Cummings Publishing Co., Inc., Menlo Park
5. Old, R.W. and Primrose, S.B. (1988), Principles of gene manipulation. An introduction to genetic engineering. Blackwell Scientific Publicatin, Oxford
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