For chromosomal painting studies we used kits produced at the Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, UK, by separation of whole chromosomes using flow cytometry (chromosomes 1–9 of Gallus gallus - GGA - and all chromosomes of Burhinus oedicnemus - BOE). From the products of the primary PCR performed to amplify the DNA of the isolated chromosomes, a second round of DOP-PCR, using 1 µl of product, allowed its labelling with Cy3-dUTP, biotin-16-dUTP (Boehringer Mannheim) or fluorescein isothiocyanate − 12-dUTP (Amersham), subsequently detected with avidin-FITC or avidin-FITC.
For the hybridization experiments, metaphase chromosome preparations were aged for 1 h at 65 °C and treated in 1 % pepsin for 5 min. Chromosomal DNA was denatured at 60 °C in 70 % formamide for 30 seconds. The probes were denatured under the same conditions. The probes were hybridized for three days at 37 °C. After that the slides were washed twice in formamide 50 %, 2xSSC, and once in 4xSSC/Tween at 40˚C. For visualization of the biotin-labelled probes a layer of Cy3-a or Cy5-avidin (1:1000 dilution; Amersham) was used. For FITC-labelled probes we used a layer of rabbit anti-FITC (1:200; DAKO). Slides were mounted in a mounting medium with DAPI called Vectashield (Vector Laboratories) [32 (link)].
For the hybridization experiments, metaphase chromosome preparations were aged for 1 h at 65 °C and treated in 1 % pepsin for 5 min. Chromosomal DNA was denatured at 60 °C in 70 % formamide for 30 seconds. The probes were denatured under the same conditions. The probes were hybridized for three days at 37 °C. After that the slides were washed twice in formamide 50 %, 2xSSC, and once in 4xSSC/Tween at 40˚C. For visualization of the biotin-labelled probes a layer of Cy3-a or Cy5-avidin (1:1000 dilution; Amersham) was used. For FITC-labelled probes we used a layer of rabbit anti-FITC (1:200; DAKO). Slides were mounted in a mounting medium with DAPI called Vectashield (Vector Laboratories) [32 (link)].
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