Dual Color Detection of Repetitive DNA Sequences in Micronuclei

With the procedure described here, which is a two-color PRINS protocol, you can localize the centromeric and pericentromeric regions of murine chromosomes. In the mouse, the centromeric satellite DNA is called minor satellite, and the pericentromeric one major satellite, in agreement with their average extension on chromosomes. All murine chromosomes, except the Y, share the same consensus sequences, therefore specific primers can be designed (9). The possibility of labeling, in a tandem fashion, the centromeric and pericentromeric regions, offer the following advantages: (1) the presence of a whole chromosome in the observed micronucleus can be confirmed (if micronucleus is labeled with both fluorescences; Fig. 2B) and (2) the frequency of preferential chromosome breakages in the wide heterochromatic pericentromeric region can be investigated by estimating the frequency of micronuclei carrying the major satellite DNA only (Fig. 2B; see Subheading 3.4.3. for interpretation of the results).

Another powerful dual-color approach consists in the labeling of telomeric and centromeric DNA sequences in the same micronucleus (Fig. 2C).

1. Follow the procedure described in Subheading 3.2., steps 1 to 8. In the meantime, prepare the second reaction mix, necessary for labeling a different repetitive DNA sequence by means of a different modified nucleotide (see Note 20) and the specific primer (Table 2). Calculate 25 pL for two slides.

2. Immerse the slides in a Coplin jar containing 0.01 MTris-HCl, pH 7.6. Incubate for 2 to 5 min (see Note 21). You are ready for the second PRINS reaction: repeat steps 4 to 8 described in Subheading 3.2.

3. The detection step is formally the same as described in Subheading 3.2., steps 9 to 15. However, the detection mix contains both fluorescein-conjugated anti-digoxigenin antibodies (1:12 v/v) and Cy3-conjugated extravidin (1:300 v/v). For two slides (60 pL) mix 5 pL of fluoresceinated anti-digoxigenin antibody, 2 pL of a 1:10 working solution of Cy3-conjugated-extravidin (final dilution 1:300), 6 pL of blocking solution, and 47 pL of PBS.

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