Single Color Detection of Repetitive DNA Sequences in Micronuclei

The procedure described here can be appliedfollowingtherationaledepicted in Fig. 2A. The centromeric region of humanchromosomescanbedetectedby means of a primer designed on the 17-base pair motif of the CENP-B box (7). This sequence is found in alphoid DNA repeats at centromeres, and it is shared by all autosomes and the X-chromosome of humans and mice (7). Therefore, the protocol described here also can be applied on murine cells (see Subheading 3.3. for a most useful protocol). With few modifications, the single-color protocol can be used for the detection of telomeric repeats in mammalian cells. In hamster, the pericentromeric regions consist of huge blocks of nonfunctional telomeric repeats (8), whereas a true centromeric probe is still not available for molecular cytogenetics. Therefore, the telomeric repeat is a marker of the pericentromeric regioninhamstercelllines.

1. Immediately before starting the procedure,preparethereactionmixasdescribed in Table 1 (see Note 5). Centrifuge briefly and keep on ice.

2. Move the slides from -20°C to room temperature, drain the excess ethanol, and allow to dry perfectly. Select under the phase contrast microscope the working area and mark its position on the backside of the slide with a diamond-tip pen.

3. Place a Coplin jar containing 50 mL of denaturating solution on ice, immerse the slides (maximum of four, see Note 15), and denature the preparations for 1 min, 30 s (see Notes 15 and 16). Rinse briefly in 50 mL of 0.01 M Tris-HCl, pH 7.6 (shake the Coplin jar gently ). Transfer the slides to fresh Tris-HCl solution and incubate for 2 to 3 min to completely stop the denaturating action of NaOH.

4. Immediately before starting the PRINS reaction, add 0.5 mL of Taq DNA polymerase (5 U/|L; see Note 6). Prewarm the reaction mixture at the annealing temperature (Table 2) and proceed immediately.

Table 1

Reaction Mix for Single-Color PRINS

Reaction Mix for Single-Color PRINS

Table 1

Stock

Final

Mix component

concentration

concentration

Volume (| L)

Buffer

10X

1X

2.5

MgCl2

25 mM

1.5 mM

1.5

(dATP, dGTP, dCTP) cocktail

5 mM

0.2 mM

1

dTTP

0.5 mM

0.02 mM

1

Digoxigenin-11-dUTP

1 mM

0.02 mM

0.5

Primera

100 |M

6 |M

1.5

H2O (milliQ)

17

Total volume

25

aSee Note 14; Table 2.

The quantities indicated are for two slides.

aSee Note 14; Table 2.

The quantities indicated are for two slides.

5. Remove one slide from the Coplinjar, drain the excess saline solution by keeping it briefly in a tilted position on blotting paper, and then wipe the backside. Place the slide on the prewarmed in situ PCR block at the annealing temperature. At this step, the cell spot must be still hydrated but not wet. Apply 12 mL of the reaction mix, cover with a 22 x 22 cover slip, and seal with nail polish (see Note 7).

6. The procedure must be repeated for all the slides as quick as possible (see Note 17), and the success of the reaction can be strongly influenced by delayed actions.

7. Start the PRINS program (Table 2).

8. In a Coplin jar, prewarm the stop solution to 65°C. At the end of the PRINS reaction, immerse the slides in the stop solution. After a few seconds, the nail polish seal will become soft, and can be removed easily with the help of fine forceps; generally, the cover slip detaches from the slide surface in the same moment. Otherwise, place the slide in the stop solution and shake gently: the cover slip soon after will float. Incubate the preparations for a further 2 to 3 min after the cover slips are removed.

9. Immerse slides for 5 min in approx 50 mL of 4X SSC, 0.1% Tween.

10. Transfer the slides to approx 50 mL of PBST and wash for 5 min.

11. In the meantime, the detection mix should be prepared, which is fluorescein-conjugated antidigoxigenin mouse antibody (Fab fragment), 1:12 v/v, 1X blocking solution, and PBS. For two slides, prepare 60 mL of detection mix; therefore, 5 mL of the anti-dig antibody, 6 mL of blocking solution, and 49 mL of PBS. Keep the mix on ice preserving from light.

12. Apply 30 mL of detection mix per slide, cover with a 24 x 24 cover slip, and place slides in a humidified chamber (see Note 18). Incubate slides for 1 h at 37°C.

13. Wash the slides three times (5 min each) in PBS. The cover slips will become detached in the first washing step by gently shaking the Coplin jar (see Note 19). Work in the dark as much as possible to avoid fluorescence bleaching.

Table 2

Primer Sequences and Reaction Specifications for PRINS in Micronuclei of Mammalian Cells

Table 2

Primer Sequences and Reaction Specifications for PRINS in Micronuclei of Mammalian Cells

Primer

Sequence

Annealing

Extension

detected

conditions

conditions

CENP-box

5'-TGAGGCCCATCGTTGGAAAAGGAAATATC-3'

Centromeric alphoid

53°C

63°C

repetitive sequences

10 min

30 min

of humans and mice

Telomere

Telomeric sequences

53°C

72°C

5'-(TTAGGG)5-3'

of humans and mice;

10 min

30 min

pericentromeric,

telomere-like sequences

of hamster

Minor

5'-GGAAAATGATAAAAACCACACTGTACAACATATTA-3'

Mouse centromeric DNA

50-53°C8

72°C

(minor satellite)

10 min

30 min

Major

5'-CACTTTAGGACGTGAAATATGGCGAGGAAAACTGA- 3'

Mouse pericentromeric DNA

50°C

63°C

(major satellite)

10 min

30 min

"The size of fluorescent spots can result too small, especially in the case of tandem labeling with the large repetitive blocks of major satellite. Adjust the annealing temperature to improve the quality of the signals according to cell type and denaturation conditions.

"The size of fluorescent spots can result too small, especially in the case of tandem labeling with the large repetitive blocks of major satellite. Adjust the annealing temperature to improve the quality of the signals according to cell type and denaturation conditions.

14. Mount the slides with 25 to 30 mL of DAPI/antifade solution under a 24 x 24 cover slip.

15. Let the excess mounting medium dry. Approximately 1 h later, you can permanently seal the slides with nail polish. Slides can be maintained at 4°C until scored. Criteria for analysis and data interpretation are given in Subheading 3.4.3.

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