Molecular Genetic Studies on Human Sarcomas

Genetic aberrations in sarcomas 10 2.1. Cytogenetic alterations 10 2.2. Molecular genetic changes 13 2.2.1. Structural alterations of cellular proto-oncogenes 13 2.2.2. Gene amplifications 13 2.2.3. Alterations of tumor suppressor genes and loss of 17 DNA sequences 2.3. Hereditary predisposition 18 3. DNA amplification 19 3.1. Structure and mechanisms of DNA amplification 20 4. Methods for detecting aberrations in DNA copy number and gene 21 expression 4.1. Comparative genomic hybridization 21 4.2. Microsatellite marker analysis 22 4.3. The array technique and cDNA microarrays 23 AIMS OF THE STUDY 2 4 MATERIALS AND METHODS 2 5 5. Materials 25 6. Methods 28 6.1. Microsatellite marker analysis (studies I-IV) 28 6.2. Southern blotting analysis (studies I and III) 29 6.3. Linkage analysis (study IV) 29 6.4. Genomic PCR and sequence analysis (study IV) 30 6.5. Restriction enzyme analysis (study IV) 30 6.6. cDNA microarray analysis (study V) 31 6.7. Reverse transcriptase polymerase chain reaction (RT-PCR) 31 (studies IV and V) RESULTS 3 3 7. Microsatellite markers as tools for the characterization of DNA 33 amplifications-applying the method to study the 12q amplicon (studies I and II) 8. Characterization of amplicon structure from the 17p region 34 by microsatellite marker analysis and Southern blotting (study III) 9. Linkage analysis and mutation detection of the EXT2 gene (study IV) 34 10. Changes in gene expression in human osteosarcoma cell lines 35 and primary tumors (study V) DISCUSSION 3 6 11. Applicability of microsatellite marker analysis in studies on DNA 36 amplifications (study II) 12. Genetic background of a Finnish family with hereditary multiple 39 exostoses (study IV) 14. Aberrant gene expression in human osteosarcoma (study V) 40 SUMMARY AND CONCLUSIONS 4 2 ACKNOWLEDGEMENTS 4 4