Wilhelm bernhard workshop on the cell nucleus

DIRECT AMPLIFICATION PRODUCTS AND ULTRASTRUCTURE ANALYSIS OF THIN CRYOSECTIONED CELLS

Falconi M., Pelotti S*, Bini C., Galanzi A.C., Ferri G.M, Teti G., Breschi L., Ceccardi S., Mazzotti G.

Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore and *Dipartimento di Medicina e Sanità Pubblica, Sezione di Medicina Legale, University of Bologna, Italy
The PCR technology and the microdissection based methods are usefull tools in diagnostic and research fields where correlation between morphology and genotype is crucial. Our previous study described at nanometric resolution level the nuclear morphology of cryosectioned cells of about 100 nm thickness using a field emission in lens scanning electron microscopy (FEISEM) which permits the detection of secondary electron without any coating allowing in situ hybridization or immunocytochemical localization. Cryosectioned cells collected on silicon chips were submitted to molecular analysis via direct amplification of amelogenin locus and hypervariable regions II (HVII) of mitochondrial DNA. The protocol was developed utilizing cryosectiones of cells fixed in formalin and methanol- acetic acid collected on silicon chips in order to couple morphological and molecular informations. The FEISEM analysis of ultrathin cryosections revealed morphological differences on citoplasmic and nuclear organization related to the different fixatives while no substantial morphological differences were observed after the DNA amplification. All the samples were successfully amplified. For amelogenin a product of 103 bp corrisponding to X chromosome locus was identified in all the samples, HVII mtDNA was sequenced; the results were compared in fixed and unfixed cells. The possibility to identify specific cell regions at nanometric resolution coupled with molecular analysis on samples that can be utilized for further investigations represents a useful approach for the complete understanding of genetic mechanism.