B

Fig. 2. Triple-color PRINS by omitting the blocking step for simultaneous detection of three different chromosomes in a normal sample using the bio-dig-bio labeling order and avidin-fluorescein/anti-dig-rhodamine detection system. The yellow signals in nuclei (A) and a metaphase (B) correspond to the first PRINS target site at the centromere of chromosome 7. The red signals were generated by the second PRINS target and correspond to the site of the centromere of chromosome 8. The pure-green signals correspond to the third PRINS site located at the centromere of chromosome 18 (8). (Please see color insert following p. 48.)

Fig. 3. Human telomere signals detected by double-strand PRINS. The number of telomere signals is very high when using the dual-color PRINS. This typical metaphase spread shows that three signal patterns can be identified. The green signal pattern was generated only from (TTAGGG)7 primer extension and labeled by biotin-dUTP, the red signal pattern was generated only from (CCCTAA)7 primer extension and labeled by digoxigenin-dUTP, and the combined green and red signal pattern (yellow) was generated from extension of both complementary primers (11). (Please see color insert following p. 48.)

6. 2X SSC (diluted from 20X SSC), pH 7.2-7.4. Store the SSC solutions at ambient temperature and discard after 6 mo or sooner if the solution appears cloudy or contaminated.

7. TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM ethylene diamine tetraacetic acid (EDTA) (see Note 1).

8. Denaturation solution: 70% formamide/2X SSC.

10. Incubator set at 37°C.

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