Methods

In essence, the PRINS method consists of 4 steps: (1) DNA denaturation, (2) annealing of primers, (3) chain extension in the presence of labeled nucleotides and Taq DNA polymerase, and (4) detection of newly-synthesized labeled DNA by use of antibodies complexed to fluorescent dyes. The oligo-nucleotide primers are 16 to 30 base pairs in length. A typical reaction mixture for PRINS has a volume of 40 |L and contains 200 to 500 pmol of primer (see Note 4). The reaction mixture is placed on the denatured chromosome preparation, with denaturation having been performed by immersing the slides in 70% formamide-2X SSC for 2 to 3 minat 72°C, followed by dehydration through a series of cold ethanol washes. The slides are air-dried, the reaction mixtures are layered onto the slides, and the slides are coverglassed and sealed. The slides are put on a flat-plate programmable thermal cycler (a water bath can be used instead) and maintained at annealing temperatures for 5 to 10 min, followed by extension for 20 to 30 min at 70-72°C. The reaction is stopped by immersing the slides in blocking solution (500 mM NaCl, 50 mM ethylene diamine tetraacetic acid, pH 8.0) for 5 min at 72°C. Labeled DNA is detected by immunocytochemistry and fluorescence microscopy. In our studies, biotin-complexed fragments are visualized by use of fluoresceinated avidin. The chromosome preparations are counterstained with DAPI and scored under ultraviolet (UV) light. The specific step-by-step procedures are listed in the subheadings to follow.

3.1. Preparing the Culture Medium

1. Mix 100 mL of fetal bovine serum (FBS) with 400 mL of RPMI-1640 in a separate bottle. Agitate well. Prepare fresh mixture every 30 d. Store at 4°C.

2. To each bottle of medium, add 3 mL of sodium heparin, 2 mL of gentamycin sulfate, and 4 mL of L-glutamine.

3.2. Initiation of Bone Marrow Culture

1. Two cultures are initiated for each patient.

2. For each culture, add 10 mL of culture medium to two 15-mL centrifuge tubes.

3. Using a Pasteur pipet, inoculate each centrifuge tube with 12 to 15 drops of cultured bone marrow cells. Each centrifuge tube represents a separate culture. Do not add mitogen. Label the tubes.

4. Incubate the cultures at 37°C for 72 h (in general the cultures may remain undisturbed for the full incubation period, although some groups gently agitate the cultures each day).

3.3. Harvesting the Cells

1. Using an 18-gage syringe needle, add five drops of colcemid (10 pg/mL) to each culture.

2. After mixing well, incubate the cultures for 1 to 2 h at 37°C. It may be necessary to increase incubation time with colcemid.

3. Centrifuge at 250^ for 10 min.

4. Remove the supernatant and break up the pellet by agitation with a vortex mixer.

5. Add 10 mL of hypotonic KCl-Na citrate mixture to each culture. Suspend the cells by gently inverting the centrifuge tubes manually. Allow the tubes to stand at room temperature for 30 min.

6. Suspend the cells again by gently inverting the tubes. Then, add 2 mL of freshly prepared fixative solution directly to each tube. Mix the contents by inverting the tubes, and centrifuge the tubes again at 250^ for 10 min.

7. Remove all of the supernatant from each culture. Gently "thump" the tubes to break up the cellular pellets.

8. Add 10 mL of fresh fixative to each culture. Resuspend the cell pellets by inverting the tubes and allow the cultures to stand at room temperature for 10 min and centrifuge.

9. Repeat the preceding step.

10. The next steps are performed in a "harvesting room" with a humidifier. For maximal chromosome spreading, adjust the humidity to 55 to 65%.

11. Set a hot plate to 65°C and check the temperature with a surface thermometer.

12. Centrifuge the cell suspension at 250^for 10 min.

13. Remove the supernatant from the centrifuge tubes and add methanol:glacial acetic acid fixative to the pellet drop by drop until the suspension becomes semiclear. The amount of fixative required will depend on the size of the pellet. The final cell concentration may have to be adjusted after evaluation of the first slide.

3.4. Preparing Slides for PRINS

1. Clean microscope slides are placed in slide tray containing deionized water. The slides are chilled in a refrigerator.

2. One cold slide at a time is removed from the slide tray and maintained in a slanting position on a stand or a device (at approximately a 10° to 20° angle).

3. Drop or place 40 to 50 pL of cell suspension on the slide. Allow the cell suspension to roll down the slide. Wipe the back of each slide and shake off excess fixative and water. Label the slides and place on a hot plate at 65°C for 2 to 3 min.

4. Store slides at room temperature for 24 h, after which the chromosomes are ready for the labeling procedure (see Note 5).

3.5. PRINS Labeling: Standard Protocol

1. Immerse slides in 0.02 NHCl for 20 min (see Note 6).

2. Immerse slides in 70% formamide-SSC, pH 7.0, for 2 min at 72°C to denature chromosomal DNA.

3. Dehydrate slides by passage through an ice-cold ethanol series, 70%, 85%, and100% EtOH, 5 min each. Air-dry.

4. Prepare reaction mixture in a final volume of 40 pL containing 50 pmol of each oligonucleotide primer (see Note 7), 0.2 mM each dATP, dCTP, and dGTP, 0.02 mM dTTP, 0.02 mM biotin-16-dUTP, 50 mMKCl, 10 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 0.01% BSA, and 2 U Taq DNA polymerase with TaqStart antibody (see Note 8). Pipet 40 pL of reaction mixture onto freshly prepared slide (see Note 9).

5. Cover the working area of the slide completely with a cover glass. Apply a thin application of rubber cement to seal the ends of the cover glass.

6. Incubate slides. Our incubations are carried out on a programmable thermal cycler equipped with a flat plate for slides (MISHA, Shandon Lipshaw; see Note 1). The program consists of one cycle of 10 min at annealing temperature with an additional 30 min at 72°C for extension. In the original study by Tharapel and Kadandale (9), 62.8°C was used for RB1 and 64.5°C for p53 (see Note 10 for general computation of annealing temperatures).

7. After extension, slides are removed from cycler, the cover glasses are removed, and the slides washed in 0.4X SSC at 72°C for 2 min to stop the reaction (see Note 11).

8. In our studies, biotin-labeled nucleotides are detected with the TSA Biotin System.

3.6. Signal Amplification Using the TSA Biotin System

1. For each test, dilute the stock solution of biotinyl tyramide 1:50 with 1X amplification diluent to prepare the working solution; 100 to 300 pL of working solution is needed for each slide.

2. After hybridization, block slides by incubation with 100 to 300 pL of TNB buffer. This may be performed for 30 min in the humidified chamber of the cycler with biotin-labeled probes: 100 to 300 |L of SA-HRP (streptavidin-horseradish peroxidase from the TSA kit) diluted 1:100 in TNB buffer. Fluorescein-labeled probes may be substituted: 100 to 300 |L of anti-fluorescein-HRP (NEN) diluted 1:250 in TNB buffer. Optimal concentrations of HRP-labeled reagents should be determined for individual laboratories.

3. Wash slides in TNT buffer with agitation three times for 5 min at room temperature.

4. Pipet 100 to 300 |L of working solution onto each slide. Maintain slides at room temperature for 5 to 10 min.

3.7. Fluorescence Microscopy and Visualization

1. To each slide, add 100 to 300 |L of streptavidin-fluorophore conjugate diluted in TNB buffer; use dilution recommended by manufacturer: streptavidin-Texas red (NEN) is used at 1:500.

2. Place slides in a humidified chamber for 30 min at room temperature.

3. Wash slides in TNT buffer, with agitation three times for 5 min at room temperature.

4. Place two drops of 4',6-diamino-phenylindole (DAPI II) on the slide for chromosome staining, and mount for microscopy. Blot excess DAPI, cover, and seal the ends of the cover glass with rubber cement. Refrigerate at 4°C for 30 to 60 min (see Notes 12 and 13).

4. Notes

1. An alternative programmable cycler, the "HYBrite Denaturation/Hybridization System," is produced by Vysis Inc. However, annealing and extension can be accomplished on thermoblocks, or even in metal containers suspended in hot water baths (3). As always temperature is critical with PRINS and must be carefully controlled.

2. In our experience, signal is increased by use of multiple primers (as many as four or five) for a single locus and by single-step annealing and extension.

3. Innis and Gelfand (10) note that at 20°C, Tris buffer has a pKa of 8.3, and A pKa of -0.021/°C. Therefore, the actual pH of Tris-HCl may vary during thermal cycling.

4. An unamplified slide without TSAG reagents and an amplified slide without primer should be included as controls for each hybridization.

5. Slides should be kept moist during the PRINS procedure. If a humidified chamber is not available, cover slides with a damp paper towel in a closed box. If a humidifier is available, maintain humidity at 55 to 65% for optimal chromosome spreading.

6. Treatment of slides with 0.02 N HCl removes loosely bound protein thereby rendering DNA more accessible to the primers.

7. For each study, the primer concentration should be optimized. New England Nuclear recommends a 10-fold reduction in "probe" (primer) concentration as optimal. This is a critically important step in PRINS, as improper concentration of probe can obviate development of the hybridization signal.

8. TaqStart monoclonal antibody binds Taq DNA polymerase, thereby minimizing nonspecific amplification and formation of primer dimers.

9. Reagents should completely cover cells or metaphase spreads on microscope slides.

10. After counting the A, C, G, and T nucleotide residues of the primers, annealing temperatures are computed by use of either of the following formulas:

where L = the length of the primer = the total number of nucleotides in the primer.

TM = 4 (G + C) + 2 (A + T) When different temperatures are obtained, the results may be averaged. In general, satisfactory annealing occurs at temperatures between 55° and 75°C. The higher temperatures increase annealing specificity.

11. Background staining is minimized by stringent washing of slides in SSC.

12. Low signal may be corrected by titration of HRP conjugate to optimize concentration, by increasing incubation time or concentration of amplification reagent, or by addition of a step to optimize penetration of reagents.

13. Background staining may be minimized by decreasing concentration of HRP conjugate or primers, by increasing endogenous peroxide quenching, by filtration of buffers, or by increasing number of washes or the length of washes.

References

1. Pellestor, F., Girardet, A., Andréo, B., and Charlieu J.-P. (1994) A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligo-nucleotide primed in situ labelling (PRINS). Hum. Genet. 94, 346-348.

2. Pellestor, F., Girardet, A., Lefort, G., Andréo, B., and Charlieu, J.P. (1995) Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y. Hum. Genet. 95, 12-17.

3. Koch, J., Hindkjœr, J., Kalvraa, S., and Blund, L. (1995) Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cytogenet. Cell Genet. 71, 142-147.

4. Gosden, J., Hanratty, D., Startling, J., Fantes, J., Mitchell, A.,and Porteous, D. (1991) Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization. Cytogenet. Cell Genet. 57, 100-104.

5. Cinti, C., Santi, S., and Maraldi, N. M. (1993) Localization of single copy gene by PRINS technique. Nuc. Acids Res. 21, 5799-5800.

6. Kadandale, J. S., Wachtel, S. S., Tunca, Y., Wilroy, R. S. Jr., Martens, P. R., and Tharapel, A. T. (2000) Localization of SRY by primed in situ labeling in XX and XY sex reversal. Am. J. Med. Genet. 95, 71-74.

7. Kadandale, J. S., Wachtel, S. S., Tunca, Y., Martens, P. R., Wilroy, R. S. Jr., and Tharapel, A. T. (2002) Deletion of RBM and DAZ in azoospermia: evaluation by PRINS. Am. J. Med. Genet. 107, 105-108.

8. Tharapel, A. T., Kadandale, J. S., Martens, P. R., Wachtel, S. S., and Wilroy, R. S. Jr. (2002) Prader Willi/Angelman and DiGeorge/velocardiofacial syndrome deletions: diagnosis by primed in situ labeling (PRINS). Am. J. Med. Genet. 107, 119-122.

9. Tharapel, S. A., and Kadandale, J. S. (2002) Primed in situ labeling PRINS) for evaluation of gene deletions in cancer. Am. J. Med. Genet. 107, 123-126.

10. Innis, M. A., and Gelfand, D. H. (1990) Optimization of PCRs, in PCR Protocols, A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J., eds.), Academic Press, Inc., San Diego CA, pp. 3-12.

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