29 Spectral Imaging Analysis of Metaphase and Interphase Cells
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12. The PCR products are precipitated in 1.2 vol isopropanol and resuspended in 1x TE buffer, and can be labeled in a random primer reaction.
B. Preparation of locus-specific probes via random primer reaction
1. Add 400 ng of DNA to water to a final volume of 14 III in a O.5-ml tube.
2. Boil tube at 100°C for 5 min, then quickly chill on ice. Add:
5 III
lOxA4 mix
6.5 III
1 mM dTTP
3.5 III
dig-ll-dUTP, FITC-12-dUTP, or Cy3-dUTP{1 mM)
20 III
2.5 x random primers
For biotin labeling add 5 III lOxdNTP (containing biotin-14dCTP), 10 III water, and 20 III 2.5 x random primers.
3. Mix well and add 1 III DNA polymerase I Klenow fragment
(40 Units/ill).
4. Incubate tube at 37°C for 2 h.
5. Add 5 III of lOx stop buffer (EDTA).
6. Store at -20°C.
C. Preparation of hybridization mixture for interphase cells analysis
Table 1 is an example of the 8-chromosomes probe set. Probes specific for
repeated DNA on chromosome 15 (CEPI5, satellite III, D15Z1, Vysis, Inc.),
chromosome X (CEPX, alpha satellite, DXZl, Vysis, Inc.), and chromosome Y (CEPY, satellite III, DYZl, Vysis, Inc.) are labeled with either a
green or red fluorochrome (Spectrum Green or Spectrum Orange, Vysis).
The probe specific for repeated DNA of chromosome 16 was prepared
from clone pHURI95. The locus specific DNA probes for chromosome
13 (YAC 900g6), chromosome 18 (YAC 945b6), chromosome 21 (YAC
141g6), and chromosome 22 (YAC 84ge9) were obtained from yeast artificial chromosome (YAC) clones. The DNAs from YACs were isolated
using PFGE and DOP-PCR. The DNAs from clones specific for chromo-