Chromosome Preparation

1. Vigorously growing roots of Vicia grandiflora.

2. Digestion mix: 2.5% pectolyase Y-23 (MP Biomedicals) and 2.5% cellulase "Onozuka" R-10 (Yakult Pharmaceutical Industry, Tokyo, Japan) dissolved in 75 mMKCl and 7.5 mMEDTA, pH 4.0. Store the mix in 200-pL aliquots at -20°C.

4. Microscope slides and cover slips (Menzel GmbH., Braunschweig, Germany).

5. Acid-cleaned slides: Working in a fume hood, prepare 100 mL of saturated solution of K2Cr2O7 in deionized water and slowly add 50 mL of concentrated sulfu-

ric acid with constant stirring. Submerse the glass slides individually into the solution and leave for at least 1 h, then wash the slides in several changes of excess water until the chromic acid is removed (the solution is no longer golden-brown). Submerse the slides into 65% HNO3 for 30 min and wash them in deionized water. Store the slides in 96% ethanol at room temperature and air-dry before use.

6. Acetic acid, glacial (ICN).

8. Liquid nitrogen.

2.2. PRINS Labeling

1. Frame-Seal incubation chambers, 15 x 15 mm, 65-pL capacity (MJ Research).

2. TaqDNA Polymerase, 5 U/pL (Promega).

3. 10 X Polymerase chain reaction (PCR) buffer: 500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100 (Promega).

4. 25 mMMgCl2 (Promega).

5. Dideoxy-nucleotide mix: 0.1 mMof each of ddATP, ddCTP, dd-GTP, and ddTTP (all Boehringer Mannheim).

6. Washing solution: 1X PCR buffer, 4 mM MgCl2 .

7. Labeling mix: 1.0 mM of each of dATP, dCTP, dGTP; 0.17 mMdTTP (Promega); and 0.08 mM Alexa Fluor 488-5-dUTP (Molecular Probes).

8. Oligonucleotide primers: VicTR-B_cons-F (5'-ATA TAA GTC TTC ARA AAA T-3'), VicTR-B_cons-R (5'-GAA GAC TTA TAT TCA CTT-3'), and VicTR-B_VG-3R (5'-TTC ACC ATA TTT TCT CAW GAT TTA TGC C-3') diluted to 4 pMin sterile deionized water.

9. 2X Standard saline citrate (SSC): 0.3 MNaCl, 0.03 M sodium citrate, pH 7.0 (sterile).

2.3. Counterstaining and Microscopy

1. Mcllvaine's buffer: 164 mM Na2HPO4, 18 mM citric acid, pH 7.0.

2. DAPI stock solution: dissolve DAPI (Sigma) in deionized water to concentration of 100 pg/mL. Filter through a 0.22-pm filter (Millipore) and store in aliquots at -20°C.

3. 4X SSC/Tween: 0.6 MNaCl, 0.06 Msodium citrate, pH 7.0, 0.2% (v/v) Tween-20 (Sigma).

4. Antifade: Vectashield mounting medium (Vector Laboratories).

5. Parafilm.

3. Methods

3.1. Chromosome Preparation

1. Excise root tips (10-15 mm long) and pretreat them in 15 pM oryzalin for 2-4 h at 25°C in the dark.

2. Remove the pre-treatment solution and fix the root tips in ethanol:acetic acid (3:1) for 2 h at 4°C and store in 70% ethanol at -20°C for up to 18 h (see Note 1).

Wash 100 root tips fixed in ethanol:acetic acid for at least 30 min in 500 mL of deionized water to remove the fixative.

Using a scalpel, remove as much of the nonmeristematic tissue as possible (i.e., remove the root cap and use only the terminal part of the root tip).

Transfer the meristems into 200 pL of enzyme solution and digest their cell walls at 27°C until the material is soft but still maintains its morphology (see Note 2).

Wash the meristems in 10 mL of 75 mMKCl for at least 15 min.

Transfer the material into 12 pL of 45% aqueous acetic acid on acid-cleaned slide (see Note 3); use three root tips for each slide.

Gently disperse the tissue, apply a clean 24- x 24-mm cover slip (see Note 4) and squash the protoplasts using a pressure adequate to restrict blood to the thumb nail. Immerse the preparation into liquid nitrogen, then flick off the cover slip with a scalpel or razor blade.

Dehydrate the preparation in an ethanol series (2 x 5 min in 70% and 2 x 5 min in 96% ethanol, respectively) and allow the slide to air-dry. Store the slides in a dessicator at -20°C (see Note 5).

PRINS Labeling

Attach the Frame-Seal chamber to the slide region containing chromosomes and incubate the slide for 5 min at 60°C to improve frame adhesion. For each slide, prepare 70 pL of PRINS mix consisting of 40.2 pL of H2O, 7 pL of 10X PCR buffer, 7 pL of labeling mix, 11.2 pL of 25 mMMgCl2, 3.5 pL of 4 pM primer (see Notes 6 and 7), and 1.1 pL of Taq polymerase. Keep the mix on ice during preparation and until application to the slide.

Dideoxy-blocking of 3'-ends (see Note 8): For each slide, prepare 100 pL of blocking mix consisting of 63 pL of H2O, 10 pL of 10X PCR buffer, 10 pL of dideoxy-nucleotide mix, 16 pL of 25 mMMgCl2, and 1 pL of Taq polymerase. Pipette the mix into the frame and cover it with a 24- x 24-mm cover slip (do not remove the top liner from the frame).

Incubate for 15 min at 72°C on a heating block using a humidity chamber (see Note 9). Equilibrate the slide to room temperature and remove the blocking mix using a pipet.

Apply 100 pL of washing solution and incubate at room temperature for 3 min. Remove the mix and repeat the washing step two more times (see Note 10). Upon completion, remove the washing solution as completely as possible. Remove the top liner from the Frame-Seal chamber, put the slide on a Petri dish chilled on ice, and pipet 70 pL of the PRINS mix at one end of the chamber. Seal the frame using polyester cover and transfer the slide onto a heating block (see Note 11) preheated to 94°C.

Immediately run the incubation profile consisting of DNA denaturation for 3 min at 94°C, primer annealing for 5 min at 45°C (see Note 12), heating to 72°C at the rate of 0.1°C/s, and primer extension for 30 min at 72°C. Remove the frame and wash the slide in 2X SSC for 3 min at room temperature.

♦ \ , + X m r *


m v^*

Fig. 1. PRINS labeling of VicTR-B repeats on Vicia grandiflora chromosomes. The reaction was performed by using primers VicTR-B_cons-F and VicTR-B_cons-R, allowing labeling of all VicTR-B sequences (A) or by using a single primer, VicTR-B_VG-3R, designed to visualize only a subfamily of the repeats (B). Note that this repeat subfamily is predominantly located in (sub)telomeric chromosome regions and is absent in other regions containing VicTR-B sequences (e.g., the prominent intercalary bands marked with arrowheads on A are not labeled on B). The chromosomes were counterstained with DAPI and appear gray, the PRINS signals are white.

3.3. Counterstaining and Microscopy

1. Dilute DAPI stock solution to 2 |g/mL in McIlvaine's buffer.

2. Add 80 |L of DAPI working solution to each slide, cover with a piece of Parafilm and incubate for 10 min at room temperature.

3. Remove Parafilm and wash slides for 5 to 10 s in 80 mL of 4X SSC/Tween in a Coplin jar.

4. Mount the preparation using a drop of antifade solution.

5. Observe the chromosomes using an epifluorescence microscope equipped with appropriate filter sets (e.g., UV-2A for DAPI and B-2A for Alexa Fluor 488) on Nikon Eclipse-600 microscope. Typical results are shown in Fig. 1.

4. Notes

1. It is very useful to synchronize cell divisions within the meristems before their fixation and chromosome preparation. Cell cycle synchronization protocols are available for a wide range of species (18).

2. The digestion takes 20 to 30 min at 27°C for Vicia meristems. However, appropriate digestion time may be different for other species and has to be determined empirically.

3. If the chromosomes and nuclei do not adhere well to the slides and are being lost during subsequent treatments, use slides coated with reagents that improve chromosome adhesion to the surface (e.g., Super Frost Plus slides, Menzel Gmbh.).

4. Do not use acid-cleaned cover slips to which chromosomes would stick.

5. The preparations can be used the next day or stored for up to several months.

6. There is only one primer used in this reaction; however, two or more primers can be used simultaneously to provide better coverage of the target sequence. In that case, the same amount of each primer (3.5 pL) is used and the volume of H2O in the mix has to be adjusted accordingly.

7. It is strongly recommended to also run a control reaction using the PRINS mix without primers. This reaction will allow clear discrimination between primer-induced and eventual unspecific background signals.

8. Pretreatment of slides with dideoxynucleotides reduces unspecific background by blocking free 3'-ends of chromosomal DNA generated during chromosome preparation. After the incorporation of ddNTPs, these ends can no longer be extended in the presence of labeled nucleotides in the subsequent PRINS reaction. Because this treatment improves signal-to-background ratio, it is especially useful for the detection of less-abundant targets that could be obscured by the background. However, in most cases, it is not necessary and steps 3 to 5 may be omitted.

9. A humidity chamber can be made by adding a piece of water-soaked tissue to the heating block equilibrated to 72°C approx 10 to 15 min before the slide incubation (it is necessary to use the block mounted within a closed chamber, see Note 11). This is required to avoid evaporation of the blocking mix as the cover slip is not tightly attached to the Frame-Seal chamber.

10. This treatment removes unincorporated ddNTPs that would otherwise block the PRINS reaction.

11. There are a variety of instruments equipped with flat block chambers suitable for slide incubation; in our laboratory, we use MJ Research PTC-200 with Twin Tower module (MJ Research).

12. Annealing temperature depends on the primer length and base composition and is usually between 45 and 65°C. The primers described in this chapter were annealed at 45°C (VicTR-B_cons-F, VicTR-B_cons-R) and 65°C (VicTR-B_VG-3R), respectively.


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