1. The age of slides is an important parameter. Slides should be used within a week of preparation. Best results in standard PRINS reaction are obtained with 2-d-old spreads because they give the best signals and chromosomal morphology. Using slides more than 1 to 2 wk old can be successful, but may lead to reduced sensitivity.

2. Several companies commercialize specialized thermal cyclers with flat block, for example, Techne Corporation (Cambridge, UK), MJ Research. (Watertown, MA), Hybaid, and Perkin Elmer. Because of differences in heat block design, technical conditions need to be optimized for the respective instrument used. Attaining accurate temperature at the top surface of the slide is crucial for PRINS

Fig. 1. Combined PRINS and PNA labeling on a chromosome spread. Chromosome 9 is labeled by PRINS in green. Chromosomes 1 and 18 are labeled by PNA probes, in blue and red, respectively. (Please see color insert following p. 48.)

reaction. Some programmable thermal cyclers, for example, the Hybaid Omnislide, possess incorporated control software to compensate for the temperature difference between the block and the surface of the slide.

3. PNA probes can be prepared after standard solid-phase synthesis protocols for peptides, but the production requires laboratories with the experience or the resources to support manual or automated peptide synthesis and consequently it is not easily accessible for cytogenetics laboratories. The commercial availability of PNA probes for cytogenetic purposes is still limited to consensus telomeric and a few human-specific satellite DNA probes. Until 2001, Boston Probes Inc. (Bedford, MA) was the leader in the development of PNA technology. In November 2001, the company was acquired by Applied Biosystems, which has pursued the development and the commercialization of PNA probes. A custom PNA probe service, PNA design guidelines, and a PNA probe order service are available on the Applied Biosystems web site ( DAKO A/S (Glostrup, Denmak), which was the majority owner of Boston Probe Inc., has always made available a commercialized consensus telomeric PNA

probe kit ( The PNA probes are compatible with a wide range of reporter molecules and fluorochromes, including fluoresceine and rhodamine, as well as cyanine and Alexa dyes available for a large variety of colors. The price of human chromosome PNA probe remains more expensive than the corresponding fluorescence in situ hybridization probes. However, one can hope that the success of the first generation of PNA probes will stimulate the future production of an extended variety of PNA probes and a decrease in their cost.

4. As an alternative, thermic denaturation can be used. In this case, apply the PRINS reaction mixture directly on the air-dried slide (after step 2 in Subheading 3.1.), under a cover slip. Seal with rubber cement, air dry the rubber cement, and place the slide on the heating block of the thermal cycler. Set up the following program: 3 min at 94°C to ensure the denaturation of chromosomal DNA, followed by the standard PRINS program (Subheading 3.2., step 3a), or the fast PRINS program (Subheading 3.2., step 3b).

5. A seal is not required for PRINS reactions using chemical denaturation. Both the volume of the mixture and the short incubation time prevent the slide from drying out during the reaction. For PRINS with thermic denaturation (Note 4) or PNA hybridization, rubber cement provides an adequate seal that is easily and completely removed at the end of the reaction. Nail polish also provides a very secure seal but is more difficult to remove at the end of the procedure. It also is possible to use adhesive plastic frames and cover slips (e.g., Hybaid Sure Seal), which allow a "window" on the surface of the slide, to which one can spread the reaction mixture and ensure a good seal during the reaction. In all cases, care should be taken not to trap any air bubbles. Bubbles (including small ones) will expand during the reaction and strongly affect the quality of the labeling by creating on the slide large area without signals.

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