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The localizations of genes or specific nucleotide sequences on a chromosome play a principal role for understanding genome structures. For this purpose, fluorescence in situ hybridization (FISH) method is popularly used for fluorescent labeling of the loci of target genes and the positions of bacterial artificial chromosome (BAC) markers on chromosomes. Scanning near-field optical/ atomic force microscope (SNOM/AFM) enables us to map FISH signals with spatially higher resolution than that on conventional fluorescence microscope (FM). We have applied SNOM/AFM to FISH observation and succeeded to separately observe FISH signals from two adjacent BAC markers, which cannot be distinguished by FM, on a silkworm chromosome in pachyten phase (see also related poster). SNOM images of the adjacent linkage markers labeled with two different color dyes by FISH were measured with suitable excitation and optical filters for dyes. However, to map the markers on the chromosome we must superimpose the two fluorescence images using the topographic images as guidance. The superimposition causes significant positional error and degrades accuracy of relative position of markers, which cannot be ignored in the separation of very adjacent markers.
In this study, we have developed a SNOM to enable us to obtain two colors SNOM images simultaneously. This simultaneous two color SNOM measurement eliminates the superimposing error from mapping of BAC linkage markers, and improve accuracy of relative positions on a chromosome. We confirmed spectral separation of FISH signals in the two colors SNOM measurement by observation of SNOM images of a silkworm chromosome fluorescently labeled with Cy3 or Alexa488 by FISH using BAC clone sequences as probes.
For a demonstration, we observed very adjacent markers on a silkworm chromosome, which were same position on a linkage map, by simultaneous two colors measurement. Observed signals in the SNOM images overlap almost each other, however the image shows deference of patterns and positions of signals clearly, enable us to evaluate the relative position of signals without error.
This research is supported by a fund from Bio-oriented Technology Research Advancement Institution.
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