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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A117
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.
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