S. Burde, G. Joss,[1] J. A. Gonzales, C. H. Coulon, L. L. Deaven and B. L. Marrone
Los Alamos National Laboratory, Life Sciences Division, LS-5 M888, Los Alamos. NM 87545
A macro was developed to run in conjunction with the popular image analysis package NIH Image to allow simultaneous determination of mapping positions of one or two separate DNA probes with respect to cytogenetic bands by dual color fluorescence in situ hybridization (FISH) and DAPI (4,6-diamidino2-phenyl-indole dihydrochloride) banding.
Chromosomes were hybridized with cosmid probes labeled with fluorescein-11-dUTP or biotin-12-dUTP detected with Streptavidin-Texas Red. Chromosomes were counterstained with DAPI, and separate images of DAPI fluorescence and each probe fluorescence were acquired using a Zeiss Axiophot microscope fitted with a Photometrics slow-scan cooled CCD camera containing a 1 K x 1 K Kodak KAF-1400 Grade 1 chip. Images were subsequently converted into rgb-color tiff stacks and contrast-stretched for analysis.
In order to allow maximal flexibility, a user-defined line along the chromosome is used for measurements. Algorithms were developed to detect the ends of the chromosome and the cytogenetic bands. Results of the analysis are presented in graphical form, comprising a display of the DAPI intensity along the chromosome, the positions of the probe(s), the locations of bands as determined by analysis of the second derivative of the DAPI intensity profile, and a standard ideogram of the chromosome for comparison.
The approach was validated and compared to visual assignment of probes to DAPI bands using the cosmid clone PYGM which has been previously mapped to chromosome 11q13.[2] Supported by US DOE (W-7405-ENG-36)
[1] School of Biological Sciences, Macquarie University, North Ryde (Sydney), NSW 2109 Australia
[2] C. Junien, V. van Heyningen, G. Evans, P. Little and M. Mannens: Report of the Second Chromosome 11 Workshop: Genomics 12: 620-25 (1992)