6th ESACP Congress, Heidelberg, April 7-11, 1999

A117
3D-GENOME NANOSTRUCTURE ANALYSIS OF THE PRADER WILLI IMPRINTED REGION BY SPECTRAL PRECISION DISTANCE MICROSCOPY (SPDM)
Rauch J 1, Bornfleth H 1, Solovei I 2, Knoch TA 3, Horsthemke B 4, Hausmann M 1, Cremer T 2, Cremer C 1

1) Institute of Applied Physics, University of Heidelberg, 2) Institute of Anthropolpgy and Human Genetics, LMU Munich, 3) German Cancer Research Center, Heidelberg, 4) Institute of Human Genetics, University Clinics, Essen, Germany

For the study of the three-dimensional (3D) structure of the human genome and its functional significance, small DNA sequences can specifically be labeled in intact cell nuclei by fluorescence in situ hybridization. For the quantitative analysis of the topology of the labeled sites, far field light microscopes of high resolution and precision are required. For this, the technique of Spectral Precision Distance Microscopy (SPDM) has been introduced. The principle is based on the fact that the intensity bary center of a labeled site can be localized with higher precision than the resolution (= full width at half maximum of the point-spread function). If different labeled sites with distances below the conventional resolution limit are discriminated by different spectral signatures, they can be localized independently and thus their distances can be determined. For a confocal laser scanning microscope, a "resolution equivalent" of about 50 nanometers (nm) can be obtained. This, however, requires a precise calibration of imaging errors, especially the chromatic shift, by means of micro beads or in situ by simultaneous multi color labeling. SPDM was applied to study the distances among selected clones of the Prader-Willi- Syndrome Region of chromosome 15. DNA probes of four different clones were investigated. Their genomic distance in kilo base pairs was known. After two-colour fluorescence in situ hybridization in human fibroblast cell nuclei, the signals were localized with high precision and their distances were determined. 3D-distances considerably below the conventional resolution limit were obtained. A comparison to experiments using one-colour labeling of both clones showed the superiority of the new method. The relation between genomic distances of the clones and their measured geometric distances (in nm) may lead to a more precise knowledge of the topology of this region.