craig-fastpaintingofhuman-1998.pdf (112.58 kB)
Fast-painting of human metaphase spreads using a chromosome-specific, repeat-depleted DNA library probe
journal contribution
posted on 1998-05-01, 00:00 authored by M Durm, L Schüssler, H Münch, Jeffrey CraigJeffrey Craig, H Ludwig, M Hausmann, C CremerFor chromosome painting, in situ suppression of repetitive DNA sequences has been well established. Such standard protocols usually require large amounts of Cot-I DNA. Recently, it has become possible to deplete repetitive DNA sequences from library probes by magnetic purification and PCR-assisted affinity chromatography. These "repeat-depleted library probes" appear to be extremely useful for Fast-FISH, a technique that omits denaturing chemical agents such as formamide in the hybridization buffer, resulting in a substantial acceleration and simplification of the complete protocol. Shown here is the application of Fast-FISH to a repeat-depleted, directly fluorochrome-labeled library probe of the q-arm of chromosome 15 (Fast-Painting) for human lymphocyte metaphase spreads. Following painting without Cot-I DNA and without formamide, visual inspection revealed sufficient chromosome painting after a few hours of hybridization. The fluorescence signals of the labeling sites were analyzed after hybridization times of 1 and 2 h (in one case, 4 h) using digital fluorescence microscopy. The painting efficiency expressed in values of relative fluorescence signal ratios was quantitatively evaluated by image analysis using line-scan procedures and area-morphometry of mean luminance. Two preparation protocols (ethanol dehydration without and with RNase A treatment followed by pepsin digestion for four different exposure times) were compared. These results indicated that RNase A treatment and pepsin digestion are steps that can be omitted.
History
Journal
BiotechniquesVolume
24Issue
5Pagination
820 - 825Publisher
Future ScienceLocation
London, Eng.Publisher DOI
Link to full text
ISSN
0736-6205Language
engPublication classification
C1.1 Refereed article in a scholarly journalCopyright notice
2018, Author(s)Usage metrics
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AdultChromosomes, Human, Pair 15Gene LibraryHumansIn Situ Hybridization, FluorescenceLymphocytesMetaphaseMicroscopy, FluorescenceMicroscopy, VideoOligonucleotide ProbesRepetitive Sequences, Nucleic AcidSensitivity and SpecificitySignal Processing, Computer-AssistedStaining and LabelingScience & TechnologyLife Sciences & BiomedicineBiochemical Research MethodsBiochemistry & Molecular BiologyIN-SITU HYBRIDIZATIONINSITU HYBRIDIZATIONCELLS
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