Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes

Complex probes used in fluorescence in situ hybridization (FISH) usually co ntain repetitive DNA sequences. For chromosome painting, in sito suppressio n of these repetitive DNA sequences has been well established. Standard pai nting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and aff inity PCR. Such a 'repeat depleted library probe' was hybridized to the q-a rm of chromosome 15 of human metaphase spreads and interphase cell nuclei w ithout any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH cond itions were used. After in situ hybridization, microscope images were obtai ned comparable to those achieved with the #15q library probe prior to deple tion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting effici ency expressed in terms of relative fluorescence signal intensity was quant itatively evaluated. The painting efficiency using the repeat depleted prob e of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a hi gh FISH efficiency. Using equivalent probe concentrations, no significant d ifferences were found for FISH with standard painting probes and repeat dep leted painting probes. (C) 2000 Elsevier Science B.V. All rights reserved.