Preparation of Labeled Probe

Probes are most conveniently prepared by the random priming method of Feinberg and Vogelstein (7,8). The DNA can be in the form of intact plasmid, lambda, cosmid, or YAC clones, or a restriction fragment isolated by agarose gel electrophoresis. In the latter case, the gel used should be cast using low-melting-point agarose, and the band should be excised in a minimum volume of gel. Three volumes of sterile distilled water are then added, and the mixture boiled for 7 min before adding it to the labeling reaction. Alternatively, the DNA can be extracted from the gel, using a variety of methods, typically "freeze-squeezing," phenol extraction, or a proprietary method such as Geneclean (see Note 5).

3.3.1. Synthesis of Biotinylated Probes by Random Priming

1. Place 5 ||L oligolabeling buffer and 1 |L 1 mM biotin-16-dUTP in a microcentrifuge tube.

2. Boil 100-500 ng DNA in 20 |L water or TE for 3 min, then add 18 ||L to the tube.

3. Add 1 |L (5 units) of Klenow fragment of DNA polymerase I and incubate the reaction at room temperature for 1 h to overnight.

4. Ethanol-precipitate the labeled DNA and resuspend it in 50 |L sterile distilled water; then, add 50 ||L 2X hybridization solution and mix well. This is sufficient probe for five slides. Any unused probe may be stored at -20°C (see Note 6).

3.3.2. Synthesis of Probes from PCR Amplified DNA

Probes can be made from DNA amplified by the polymerase chain reaction

(PCR), using additional PCR cycles in the presence of a biotinylated nucleotide.

1. Remove unincorporated nucleotides from the amplified DNA (e.g., by gel elec-trophoresis).

2. Set up the PCR using the same conditions used to amplify the probe DNA, but with the following changes. Substitute biotin-16-dUTP for the TTP in the PCR. Five cycles of synthesis are usually sufficient. Increasing the length of the polymerization step to 10 min is advised, because the concentration of biotin-16-dUTP is low.

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