Syllabus
Day 1
- introduction
- preparation of mitotic chromosomes from root tip meristems (Nicotiana, Festuca ´ Lolium hybrids) and preparation of meiotic chromosome spreads from anthers (Arabidopsis thaliana)
- theoretical and practical principles of light and fluorescence microscopy
- analysis of chromosome preparations using fluorescence microscopy (selection of suitable slides for Day 2)
- direct and indirect labelling of DNA probes (amplification of the Arabidopsis-type telomere repeat by PCR; labelling of ribosomal DNA repeats, chromosome-specific BAC clones, and genomic DNA using nick translation)
Day 2
- fluorescence in situ hybridization (FISH), chromosome painting, genomic in situ hybridization (GISH): PART I (slide pretreatment, preparation of hybridization mix, probe denaturation)
- stadia of meiosis (meiosis analyzed using phase and fluorescence microscopy, digital microscope photography)
Day 3
- FISH, chromosome painting, GISH: PART II (fluorescence detection of hybridized DNA probes)
- analysis of microscope preparations using fluorescence microscopy, image capture
- computer processing of microscope images (Adobe Photoshop)
- conclusions
Annotation
This practical course will demonstrate classical as well as state-of-the-art cytogenetic techniques. The accent is put on cytogenetic methods beneficial to taxonomic, biosystematic and phylogeographic studies.
Students will get acquainted with preparation of mitotic and meiotic chromosomes, chromosome counting, the course of male meiosis as well as with principles of light and fluorescence microscopy. Molecular cytogenetic techniques will include DNA probe labelling, fluorescene in situ hybridization (FISH), genomic in situ hybridization (GISH) and chromosome painting.
Digital processing of captured chromosome images will be shown.