Basic Ingredients and Instrumentation

1. Chromosome spreads (preferably freshly made; see Note 1).

2. Cover slips.

3. Coplin jars or other suitable containers for washing the slides.

4. Standard nucleoside triphosphates, dideoxy nucleotides (Lithium salt, e.g., Roche).

5. Hapten- or fluorochrome-labeled nucleotides (digoxigenin-2'-deoxyuridine 5'-triphosphate [dUTP], biotin-dUTP, fluorescein-dUTP, rhodamine-dUTP, etc.; e.g., Roche).

7. Thermostable DNA polymerase that will accept labelled nucleotides (e.g., Tth or Taq DNA polymerase) and 10X polymerase buffer (enzyme and buffer are supplied together by Roche and most other suppliers). For convenience, the 10X buffer may be mixed with an equal volume of glycerol into a 5X buffer that will not freeze at -20°C.

Fig. 1. ddPRINS on a spread of human chromosomes. Each chromosome is made of two chromatids, and each chromatid has two ends, so the number of targets/signals/cell is in principle 184. However, signals on neighboring chromatids may appear fused. The outline of the chromosomes is also visible, mainly owing to autofluorescence from the chromatin.

9. 500 mMEthylene diamine tetraacetic acid (EDTA).

10. 20X Standard saline citrate (SSC).

12. Ethanol.

13. Nonfat dry milk (powder).

14. Fluorochrome labeled anti-digoxigenin for detection of digoxigenin labeled PRINS product (Fab fragment, Roche).

15. Fluorochrome-labeled streptavidin for the detection of biotin-labeled PRINS product (Roche, Vector Laboratories).

16. Antifade solution: Vectashield (Vector Laboratories) or p-phenylenediamine-dihydrochloride (Sigma).

17. Counterstain: either propidium iodide (Sigma) or DAPI (Sigma).

18. If denaturation and hybridization are performed consecutively, either a special PRINS/in situ PCR machine (from Hybaid, MJ-Research or Techne) or two incu bators (thermoblocks, waterbaths) are needed. They must provide a metal surface to ensure good heat transfer so that denaturation at 94°C and hybridization at 55-65°C can be obtained. To ensure the correct temperature, it may be helpful to cover the slide and the hot plate with an insulating lid. If slides are already denatured, one incubator is sufficient.

19. Fluorescence microscope with standard excitation and emission filters (e.g., 81000 filter set from Chroma Technology).

2.2. Mixtures

1. Ice cold (-20°C) ethanol series (70%, 90%, and 99% v/v) for PRINS on predenatured slides.

2. 10X dNTP for dideoxy-PRINS (with ddGTP for telomere staining with the Telo2 primer (CCCTAA7 [1,2,8,9]): 1.0 mM each of dATP, dCTP; 1.0 mM ddGTP; 0.1 mM of labeled dUTP; mix in 50% glycerol. Can be stored at -20°C for 1 yr (see Note 3).

3. Reaction mixture (60 pL total): 6 pL 10X buffer (or 12 pL 5X), 6 pL of 10X dNTP, 1 pL (approx 1 pg) oligonucleotide, 1 pL (approx 1U enzyme), and 38 pL (or 33 pL) ultrapure water. The polymerase should not be added until the mixture is actually used, but the mixture without polymerase may be prepared weeks in advance and stored at -20°C.

4. Stop buffer: 50 mMNaCl, 50 mM ethylene diamine tetraacetic acid, pH 8.0. This buffer can be stored at room temperature for months.

5. Wash buffer: 4X SSC, pH 7.0 (1X SSC: 150 mM NaCl, 15 mM sodium citrate), 0.05% Tween-20. This buffer can be stored at room temperature for months.

6. Blocking solution: 5% (w/v) nonfat dry milk dissolved in washing buffer. Centrifuge for 2 min in an Eppendorf centrifuge and use supernatant. This solution can be stored at -20°C for years (see Note 4).

3. Methods

3.1. Pretreatment of Samples

1. The main limitation to the intensity of the signal is the extent of the chain elongation in situ. Often this stops after 150 to 200 bases, presumably as a result of increased tension in the template DNA as the DNA synthesis progresses. The increase in tension may be prevented or reduced if the preparation also is treated with an enzyme that breaks the DNA at defined positions without ruining the template, for example, a rare cutting restriction enzyme like SaH, may increase signal intensity up to 10-fold. For this pretreatment, the slide should be incubated with 1 U restriction enzyme for 1 h at the temperature optimum of the enzyme. It may be necessary to seal the cover slip to the slide because glycerol cannot be included to prevent evaporation (glycerol enhances DNA polymerases and exo-nucleases without apparent side-effects but induces star activity of restriction enzymes). After the pretreatment, the slide should be dehydrated in an ethanol series (70%, 90%, and 99% for 3 min each), after which it is removed from the

99% ethanol, drained, and air-dried. This pretreatment has no visible effect on the telomere staining, presumably because the template DNA is at the end of the chromosome anyway but may be useful for the detection of internal repeats.

2. The target DNA can be denatured as part of the PRINS proper according to the protocol in Subheading 3.2. Alternatively, the slide may be denatured before the reaction (e.g., in 50-70% formamide, at 70°C, for 2 min). It is important that the slide is immediately quenched in a -20°C ethanol series (70%, 90%, and 99% for 3 min each) to fix the target DNA in the denatured configuration. After the dehydration, the slide is removed from the 99% ethanol, drained, and air-dried. Step 2 in the protocol is then bypassed for the PRINS proper.

3.2. PRINS Proper

1. Decide how large a region of the slide should be analyzed and choose a cover slip and an amount of reaction mixture that fits the area. Use 1-pL reaction mixture for each mm of cover slip length. To cover a standard slide completely, prepare a 60-pL reaction mixture and cover with a 24- x 60-mm cover slip. To cover a smaller area, prepare less reaction mixture (and use a smaller cover slip).

2. As soon as the reaction mixture has been spread with the cover slip, denature the slide by placing it on a hotplate covered with a lid for 4 min at 94°C (with the automated incubators the temperature could be a bit lower because they often operate from a simulated slide function, and the temperature inside the slide is slightly lower than the surface temperature of the incubator).

3. Either lower the temperature to between 55 and 65°C or transfer the slide to this temperature and incubate for 5 to 60 min for probe annealing and chain elongation (see Note 5).

4. Place the slide in preheated stop buffer at the temperature used in step 3 for 1 min to terminate the PRINS reaction.

5. Transfer the slide to 50 mL of wash buffer and wash for approx 3 min at room temperature.

The reaction can be paused at this step and the slide stored overnight in wash buffer at 4°C.

6. If fluorochrome-labeled nucleotides have been used; the slide can now be coun-terstained, mounted, and evaluated under the microscope. If digoxigenin- or biotin-labeled nucleotides have been used, they need to be visualized with anti-digoxigenin antibody or streptavidin.

3.3. Visualization of Digoxigenin- or Biotin-Labeled PRINS Products

1. Apply 50 pL of fluorochrome-conjugated antidigoxigenin (or fluorochrome-conjugated streptavidin) in blocking solution (2 ng/pL) to the slide. Incubate under a cover slip for 30 to 60 min in the dark.

2. Wash twice for 5 min in 50 mL of washing buffer. The slide is now ready for counterstaining and mounting.

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