A versatile image analysis approach for simultaneous chromosome identification and localization of FISH probes

Modern cytogenetic techniques, such as comparative genomic hybridization (C GH) and the multi-color fluorescence in situ hybridization (FISH) technique s of multiplex fluorescence in situ hybridization (M-FISH) and spectral kar yotyping (SKY), require a coordinated banding analysis to maximize their us efulness. All of the methods currently used, including Giemsa (G-) banding, Alu banding, and 4',6-diamidino-2-phenyl-indole (DAPI) banding, have serio us drawbacks. A simple and effective method to band chromosomes concurrentl y with FISH is needed. To address this problem, we stained chromosomes with DAPI and chromomycin A3, and then used an image analysis program to genera te banding by dividing the image taken with a DAPI excitation filter by the image taken with a chromomycin A3 excitation filter. The result was a meta phase spread in which the chromosomes possessed a banding pattern character istic of R-banding. The image analysis program was then used to generate li nescans of pixel intensity versus relative position along the length of chr omosomes that were banded using this technique, which we have called DIC R- banding. Each chromosome in a genome was represented by a characteristic sc an profile, which was unaffected by FISH signals. Reference linescans were prepared by karyotyping D/C R-banded chromosomes for a given species, and t hen drawing lines along the length of the known chromosomes. The linescans were combined into a spreadsheet database, which was linked by dynamic data exchange to the image analysis program and normalized for length and inten sity. The linescan of an unknown chromosome was then transferred to the spr eadsheet, where it was normalized for length and intensity and overlaid on the linescans of each chromosome in the genome. Unknown chromosomes were id entified by comparison of their graphs with graphs in the standardized refe rence genome. We have used this approach to create reference linescan karyo types of several species, and to identify chromosomes on which FISH was per formed.