After successfully attending and absolving this course students will (i) have theoretical and practical understanding of basic nucleic acid research and analysis techniques, (ii) understand basic technical terminology used in laboratory work, (iii) be able to relate theoretical knowledge of biochemistry with practical knowledge of this course in order to comprehend biological processes and their technical application in laboratory in order to (iv) understand and master specific molecular biology techniques which they will encounter later in their studies and professional careers, and lastly, but not least important (v) learn to present the results of their work to their colleagues and other experts.
1. Isolation of DNA from different tissues (1h lect. + 4 h pract.)
2. Isolation of RNA (1h lect. + 4 h pract.)
3. Quantification, concentration and purification of nucleic acids, with the emphasize on plant, phytoplasmal and viral nucleic acids. (1h lect. + 1 h pract.)
4. Polymerase chain reaction (PCR) (1h lect. + 2 h pract.)
5. nested PCR and real-time PCR (1h lect. + 3 h pract.)
6. Reverse transcriptaze-PCR (RT-PCR), random amplified polymorphic DNA (RAPD) and other similar methods (1h lect. + 4 h pract.)
7. Digestion with endonucleases and restriction fragment length polymorphism (RFLP) (1h lect. + 2 h pract.)
8. Agarose and polyacrilamide gel electrophoresis(1h lect. + 2 h pract.)
9. Southern blotting(1h lect..)
10. Northern blotting and other hybridization techniques; DNA sequencing(1h lect.)
11. Plasmids and marker genes, genetic engineering on plants(1h lect.)
12. Phytoplasmas and application of the above mentioned techniques in detection and classification of these pathogens (practical training: total nucleic acids isolation, nested polymerase chain reaction, agarose gel electrophoresis) (1h lect. + 3 h pract.)
13. Hypoviruses and application of above mentioned techniques in detection and classification of hypoviruses (practical training: total nucleic acid isolation, isolation of replicative viral RNA (dsRNA), reverse transcriptaze PCR (RT-PCR), digestion with endonucleases, restriction fragment length polymorphism (RFLP), electophoresis) (1h lect. + 3 h pract.)
14. Analysis of the results (2h lect./pract.)
- Ausubel F M. et al. Current Protocols in Molecular Biology. Wiley, New York 1994.
- Sambrook J. et al. Molecular cloning: a laboratory manual 2nd ed. CSH Laboratory press, Cold Spring Harbor 1989
- Web presentation of lectures, Biološki odsjek, PMF (http://www.pmf.unizg.hr/biol)
- Web presentation of practicals, Biološki odsjek, PMF (http://www.pmf.unizg.hr/biol)
- Allemann C. et al. (1999): Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe assessed using restriction fragment length polymorphism (RFLP) markers. Mol. Ecol. 8: 843-854.
- Ćurković Perica M. et al. (2007): Effect of indole-3-butyric acid on phytoplasmas in infected Catharanthus roseus shoots grown in vitro. FEMS Microbiol Lett 268: 171-177.
- Lee I-M. et al. (1994): Use of mycoplasmalike organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed MLO-infections in a single host plant. Phytopathology 84: 559-566