In the three-color PRINS procedure, three sequential PRINS reactions are performed, each labeling one specific chromosome. The following labeling order is used (see Note 5):
1. FITC for the first targeted chromosome.
2. TRITC for the second targeted chromosome.
3. FITC for the third targeted chromosome.
In the four-color procedure, this labeling order is supplemented with a fourth
PRINS reaction using Cascade Blue (see Fig. 1).
1. Prepare a reaction mixture in a final volume of 50 ||L containing: 0.2 mMdATP, dCTP, and dGTP, 0.02 mM dTTP, 0.02 mM FITC-12-dUTP, 50 mMKCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.01% BSA, 200 pmol of oligonucleotide primer; and 2.5U of Taq DNA polymerase. In practice, mix in a sterile microcen-
trifuge tube: 1 pL of each 1:10 diluted dATP, dCTP, and dGTP; 1 pL of the 1:100 diluted dTTP; 1 pL of FITC-12- dUTP; 1 pL of BSA; 5 pL of 10X Taq buffer; 0.5 pL of the Taq DNA polymerase; 4 pL of the specific primer (for instance, the primer specific for chromosome 1 according to the procedure illustrated in Fig. 1); and distilled water to 50 pL.
2. Place the reaction mixture under a 22 x 32 cover slip on the denatured slide, and transfer to the heating block of the thermal cycler.
3. Set up the PRINS program and start the reaction. The program consists of a unique 5-min step at the specific annealing temperature of the primer involved for both in situ annealing and elongation (see Note 6).
4. While this first reaction is running, preparethereactionmixtureforthesecond PRINS reaction as described previouslybutinsteadincorporatethespecific primer for the second targeted chromosome (for instance, chromosome 9 according to Fig. 1), and TRITC-6-dUTP.
5. On completion of the program, carefully remove the cover slip from the slide.
6. Wash the slide twice for 2 min at room temperature in 1X PBS.
7. After draining the excess 1X PBS off the slide, and before the slide is completely dry, put the second PRINS reaction mixture on the slide, and cover with a 22 x 32 cover slip.
8. Place the side again on the plate of the thermal cycler.
9. Set up the program for the second PRINS reaction: 5 min at the annealing temperature specific to the second primer used.
10. Start the program.
No additional denaturation is required after the first PRINS reaction because the sperm DNA remains denatured through the PRINS incubations.
11. While this second reaction is running, prepare the reaction mixture for the third PRINS reaction, incorporating the specific primer for the third targeted chromosome (for instance, chromosome 16 according to Fig. 1) and FITC-12-dUTP.
12. At the end of the second reaction, remove the cover slip from the slide and repeat the washing, steps 6 and 7.
13. Before the slide is completely dry, put the third PRINS reaction mixture on the slide, and cover with a 22 x 32 cover slip.
14. Place the slide on the thermal cycler.
15. Set up the program for the third PRINS reaction: 5 min at the annealing temperature specific to the third primer used.
17. At the end of this third reaction, the slide is transferred to 4X SSC, 0.05% Tween-20 for two washes (3 min each) at room temperature, with gentle agitation.
In the four-color procedure, the third reaction is followed by a fourth reaction with the primer specific for the fourth targeted chromosome (for instance, chromosome 18 as indicated in Fig. 1). No additional denaturation is needed.
1. Prepare the reaction mixture for the fourth PRINS reaction, incorporating the specific primer for the fourth targeted chromosome and Cascade Blue-7-dUTP.
2. At the end of the reaction, remove the cover slip from the slide.
3. Transfer the slide in 4X SSC, 0.05% Tween-20 for two washes (3 min each) at room temperature, with gentle agitation.
3.3. Detection and Microscopy
1. Drain the excess washing solution off the slide.
2. Mount the slide in Vectashield antifade solution containing either DAPI (0.3 pL/mL) or a mix of propidium iodide (0.3 pL/mL) and DAPI (0.3 pL/mL).
3. Cover with a 22 x 40 cover slip and seal the cover slip with rubber cement.
4. Examine the slide under the epifluorescence microscope, preferentially using first the triple or double band-pass filter, and confirming the coloration of the fluorescent spot with single band-pass filters. Figure 2 shows some typical results obtained on sperm nuclei.
5. For aneuploidy estimate in sperm samples, strict scoring criteria must be used (see Note 7).
6. The slide may be stored in the dark at 4°C for several months. 4. Notes
1. Human chromosome-specific primers are oligonucleotides, typically 18 to 35 bases long, specific for a-satellite DNA sequences. They are determined in the a-satellite DNA sequences of each chromosome. Several specific primers can be defined for the same chromosome. The concentration of the appropriate primer must be experimentally determined. Usually 200 pmol per slide in 50-pL reaction mixture is optimal.
2. The spreading of spermatozoa on the slide must be homogeneous to facilitate the screening of the preparation and to limit incorrect scoring when performing chromosomal analysis on sperm nuclei. It is particularly important to avoid both aggregation and excessive dilution of cells on the slide. A density of 70-100 spermatozoa per field under a x40 objective is optimal. Consequently, the washing procedure is a critical step. It must be performed taking into account the quality of the initial sperm sample, that is, the viscosity after liquefaction, the sperm concentration, the agglutination of spermatozoa, and the presence of cellular debris or various cell types (leukocytes, epithelial cells, immature germinal cells, bacteria). In the case of low-quality sperm samples, one or two additional washes by centrifugation may be required to obtain adequate smeared sperm slides.
3. The age of the slides is critical for the success of the PRINS reaction. In our experience, the best results are obtained with 2-d-old spreads. Sperm suspensions can also be stored in fixative at -20°C for several months, and fresh slides made by centrifuging to collect a pellet, resuspending the pellet in fresh fixative, and dropping the suspension on clean slides, which will be aged 2 d at room temperature. Using slides that are older than 1 wk can be successful, but leads to reduced labeling sensitivity.
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