Roman Hobza: Genetic Engineering

Semestral course (autumn semester, 2/2)

Mendel University of Agriculture and Forestry, Faculty of Agronomy, branch Plant Biotechnology

Structure of plant genome
1. Complexity of plant genome, nuclear and extranuclear DNA
2. Organization of plant chromosome
3. Coding and non-coding DNA
4. Repetitive DNAsequences, transposons
General principles of manipulation with nucleic acids
1. Isolation of DNA and RNA
2. Separation of DNA and RNA: centrifugation, electrophoresis
3. Hybridization of nucleic acids
4. Enzymes in genetic engineering
5. Methods of DNA sequencing
Polymerase chain reaction (PCR)
1. Principle
2. Applications: TR-PCR, RAPD, RACE-PCR, AFLP
3. Applications: diagnostics, forense analysis
DNA cloning
1. Type of vectors: plasmids, phages, artificial chromosomes
2. Techniques of DNA delivery to cells
Gene transfer into plants
1. From DNA discovery to first transgenic plants
2. Agrobacterium tumefaciens
3. Ti-plasmid and T-DNA
4. T-DNA transfer into plant cells
Transgenic plants
1. Resistance against herbicides
2. Resistance against herbivorous insects
3. Resistance to viruses
From genes to genomes and back
1. Genetic versus physical mapping
2. Sequencing of genomes
Techniques of cytogenetics
1. Isolation of chromosomes – flow cytometry and microdissection
2. Fluorescence in situ hybridization (FISH)
Construction of DNA libraries
1. cDNA and genomic libraries
2. Types of libraries depending on a type of vector
3. Application of DNA libraries
Isolation of genes
1. Forward and reverse genetics
2. Application of bioinformatics in study of gene structure and function
Production of proteins in transgenic organisms
1. Markers of transgene expression
2. Animals as bioreactors
3. Modification of physiological processes – golden rice
4. Silencing of transgenes
RNA interference
1. siRNA and miRNA
2. RNAi and gene knock-out
3. Transcriptional and post-transcriptional gene silencing