Article

Visualization, analysis, and design of COMBO-FISH probes in the grid-based GLOBE 3D genome platform.

Biophysical Genomics, Dept. Cell Biology & Genetics, Erasmus MC, Dr. Molewaterplein, Rotterdam, The Netherlands.
Studies in health technology and informatics 01/2010; 159:171-80.
Source: PubMed

ABSTRACT The genome architecture in cell nuclei plays an important role in modern microscopy for the monitoring of medical diagnosis and therapy since changes of function and dynamics of genes are interlinked with changing geometrical parameters. The planning of corresponding diagnostic experiments and their imaging is a complex and often interactive IT intensive challenge and thus makes high-performance grids a necessity. To detect genetic changes we recently developed a new form of fluorescence in situ hybridization (FISH) - COMBinatorial Oligonucleotide FISH (COMBO-FISH) - which labels small nucleotide sequences clustering at a desired genomic location. To achieve a unique hybridization spot other side clusters have to be excluded. Therefore, we have designed an interactive pipeline using the grid-based GLOBE 3D Genome Viewer and Platform to design and display different labelling variants of candidate probe sets. Thus, we have created a grid-based virtual "paper" tool for easy interactive calculation, analysis, management, and representation for COMBO-FISH probe design with many an advantage: Since all the calculations and analysis run in a grid, one can instantly and with great visual ease locate duplications of gene subsequences to guide the elimination of side clustering sequences during the probe design process, as well as get at least an impression of the 3D architectural embedding of the respective chromosome region, which is of major importance to estimate the hybridization probe dynamics. Beyond, even several people at different locations could work on the same process in a team wise manner. Consequently, we present how a complex interactive process can profit from grid infrastructure technology using our unique GLOBE 3D Genome Platform gateway towards a real interactive curative diagnosis planning and therapy monitoring.

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    ABSTRACT: With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.
    International Journal of Molecular Sciences 01/2010; 11(10):4094-105. · 2.34 Impact Factor

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