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Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes
journal contribution
posted on 2000-07-10, 00:00 authored by J Rauch, D Wolf, Jeffrey CraigJeffrey Craig, M Hausmann, C CremerComplex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting 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 affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.
History
Journal
Journal of biochemical and biophysical methodsVolume
44Issue
1-2Pagination
59 - 72Publisher
ElsevierLocation
Amsterdam, The NetherlandsPublisher DOI
ISSN
0165-022XLanguage
engPublication classification
C1.1 Refereed article in a scholarly journalCopyright notice
2000, Elsevier Science B.V.Usage metrics
Categories
Keywords
Cell NucleusChromatography, AffinityChromosomes, Human, Pair 15DNA ProbesGene LibraryHumansIn Situ Hybridization, FluorescenceMicroscopyMicroscopy, VideoPolymerase Chain ReactionRepetitive Sequences, Nucleic AcidStreptavidinFluorescence in situ hybridizationChromosome paintingRepeat-depleted probesImage analysisScience & TechnologyLife Sciences & BiomedicineBiochemical Research MethodsBiochemistry & Molecular BiologyBiophysicsINSITU HYBRIDIZATIONHUMAN-CHROMOSOMESCYTOGENETIC ANALYSISPAINTING PROBESFISHOPTIMIZATIONSEQUENCESMETAPHASEPlant Biology
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