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Extra info for Animaux en tissu : Techniques et modèles

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2. DIRECTLY LABELED PROBES 1. Probe preparation: a. Allow whole chromosome painting (WCP) hybridization buffer (Vysis) to warm to room temperature so that its viscosity decreases to the point that it may be accurately pipetted. b. In a microcentrifuge tube add 7 μL WCP hybridization buffer, 1 μl WCP DNA probe, and 2 μl deionized water. This quantity of probe mixture is sufficient to cover one 22 mm × 22 mm hybridization zone. c. To screen samples for two WCP probes simultaneously, prepare probe mixture as follows: 7 μL WCP hybridization buffer, 1 μL spectrum orange WCP DNA probe, 1 μL spectrum green WCP DNA probe, and 1 μL deionized water.

2). Because the targets are large and repeated many times, these probes generate large signals. 2. Sequence-Specific Sequence Probes The detection of unique single-copy genes (see Fig. 3) is accomplished by the use of sequence-specific probes. Levels of detection range from sequences as small as 1 kb up to as large as 300 kb (26). The various FISH unique-sequence probes are usually employed to detect microdeletion syndromes and rearrangements of oncogenes. Subtelomeric probes are produced from unique sequences in close proximity to the ends of chromosomes and are often used for the analysis of cryptic translocations.

The use of FISH based on painting and cosmid probes also requires knowledge regarding the exact anomalies to be ascertained. Detection sensitivity for FISH and other techniques is shown in Table 1. FISH Analysis 21 Table 1 Comparison of FISH with Other Assays Technique Routine pathology Cytogenetics FISH Gene rearrangement FACS analysis Clonogenic culture PCR Marker Detection limits Cellular morphology Chromosome morphology Chromosome structure DNA configuration Antigen profile In vitro growth DNA/RNA structure 10–1–10–2 10–1–10–2 10–2 10–2–10–3 10–3 10–5 10–5 FACS, fluorescence activated cell sorting; PCR, polymerase chain reaction.

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